Conjugates of an EPO Moiety and a Polymer

ABSTRACT

Conjugates of an EPO moiety and one or more non-peptidic water-soluble polymers are provided. Typically, the non-peptidic water-soluble polymer is poly(ethylene glycol) or a derivative thereof. Also provided are compositions comprising such conjugates, methods of making conjugates, and methods of administering compositions comprising such conjugates to a patient.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of and claims benefit ofpriority from U.S. patent application Ser. No. 11/357,936 filed on 16Feb. 2006. This application also claims the benefit of priority toProvisional Patent Application No. 60/653,451, filed Feb. 16, 2005. Thecontents of both of these applications are hereby incorporated herein byreference in their entirety.

FIELD OF THE INVENTION

Among other things, one or more embodiments of the present inventionrelate generally to conjugates comprising an EPO moiety (i.e., a moietyhaving erythropoietin activity) and a polymer. In addition, theinvention relates to (among other things) compositions comprisingconjugates, methods for synthesizing conjugates, and methods fortreating patients by administering such conjugates.

BACKGROUND OF THE INVENTION

One important function of the human hematopoietic system iserythropoiesis, which is the production of red blood cells or“erythrocytes.” Erythropoiesis is vitally important as it is the processwhereby new red blood cells are generated as old red blood cellsdegenerate. In this way, a continuous supply of red blood cells isensured, thereby guaranteeing the continuous oxygenation of biologicaltissues.

In humans, erythropoiesis occurs in the presence of a protein hormonecalled “erythropoietin” abbreviated as “EPO” which serves to stimulatethe division and differentiation of progenitor cells located in bonemarrow. Over a period of about four days, progenitor cells mature intored blood cells and are released into the general circulation. A typicalred blood cell lives for about four months in the systemic circulation.Typically, control of the erythropoietic cycle is governed by a negativefeedback mechanism whereby the increased oxygenation of the tissuescaused by increased numbers of red blood cells results in a decrease inthe production of erythropoietin.

Naturally-occurring EPO is a glycoprotein hormone with 165 amino acids,4 glycosylation sites on amino-acid positions 24, 38, 83, and 126, and amolecular weight of about 34,000. EPO is produced in vivo as a precursorprotein with a signal peptide of 23 amino acids and can occur in threeforms. The DNA sequences encoding EPO have been reported. See, forexample, U.S. Pat. No. 4,703,008.

Pharmacologically, EPO has been administered to humans for the treatmentof a variety of conditions, including the treatment of patientssuffering from anemia. Specifically, for example, EPO is indicated for(a) anemia associated with chronic renal failure, (b) anemia related tozidovudine therapy in HIV-infected patients, and (c) anemia in cancerpatients undergoing chemotherapy. In addition, EPO has been used toreduce allogeneic blood transfusion in patients undergoing surgery, andfor pruritus associated with renal failure.

One drawback associated with current forms of EPO therapy is thefrequency of dosing. Because EPO therapy typically requires dailyinjections, patients dislike the inconvenience and discomfort associatedwith this regimen.

One proposed solution to these problems has been to provide a prolongedrelease form of EPO. For example, U.S. Pat. No. 5,416,071 describes awater-soluble composition comprising EPO, hyaluronic acid, and humanserum albumin. Such compositions, however, may not provide sufficientactivity over the desired period of time.

Others have suggested the use of PEGylation technology, or theattachment of a poly(ethylene glycol) derivative to a protein such asEPO, in order to prolong EPO's in vivo half-life. For example, U.S.Patent Application Publication No. 2004/0082765 describes conjugation ofPEG derivatives bearing a succinimidyl lower fatty acid ester (such as aPEG derivative bearing succinimidyl propionate or succinimidyl butyratemoiety).

U.S. Patent Application Publication No. 2002/0081734 describes preparinga mutein of EPO having a cysteine residue introduced at thethirty-eighth position and then PEGylating the mutein at the introducedcysteine residue via a PEG-maleimide derivative. No specific structureof the PEG-maleimide derivative was described in the publication.

U.S. Pat. No. 6,753,165 describes methods for making soluble proteins(including EPO) having free cysteines. The publication also describesmodifying the soluble proteins by attaching a PEG moiety at the freecysteine via a PEG derivative bearing a vinylsulfone, maleimide oriodacetyl moiety. With respect to attaching the PEG, the publicationdescribes the necessity for performing a partial reduction step in orderto increase the relatively unreactive free cysteine on the protein.International Patent Publication WO 90/12874 also describescysteine-added variants of EPO, wherein a PEG is attached to the addedcysteine residue.

International Patent Publication WO 01/76639 describes myelopoietinconjugates, which refers to a fusion protein prepared from a modifiedhuman IL-3 polypeptide sequence linked to another molecule such as EPO.

U.S. Pat. No. 6,077,939 describes a process for attaching a PEG to theN-terminal alpha carbon of a protein (such as EPO) that has previouslybeen subjected to a transamination reaction. The described conjugatescan contain a PEG linked to EPO via a hydrazone, reduced hydrazone,oxime, or reduced oxime linkage.

U.S. Pat. No. 6,340,742 describes PEG-EPO conjugates wherein EPO ismodified by the addition of from 1 to 6 glycosylation sites andcovalently linked to from one to three lower alkoxy poly(ethyleneglycol) groups, each poly(ethylene glycol) group attached via a specificlinkage. The described conjugates are prepared by first “activitating”the EPO by covalently linking one or more protected thiol groups to EPO,followed by removal of the group protecting the thiol. Once theprotecting group is removed, the step of attaching certain reagents atthe unprotected thiol is performed.

U.S. Patent Application Publication No. 2003/0191291 describes proteinshaving EPO activity that are prepared using non-recombinant technology.The publication further describes such proteins that arepolymer-modified in a defined manner.

U.S. Patent Application Publication No. 2002/0115833 describes an EPOglycoprotein covalently linked to one poly(ethylene glycol) group by wayof a specific linkage containing an amide bond with the N-terminalalpha-amino group of the EPO glycoprotein.

EP 0 714 402 describes conjugates of polymers with proteins such as EPO.The poly(ethylene glycol) polymers used to form the conjugates havemolecular weights of up to about 10,000.

International Patent Publication WO 96/40792 describes conjugatesprepared from a polymer comprising the following structure:Poly(—O—C═O—Y)_(m), wherein Y is a halogen or nitrile, m is an integerfrom 1 to 25, and Poly defines a synthetic or a naturally occurringpolymer.

Notwithstanding these described conjugates, however, there remains aneed to provide conjugates or compositions of EPO that satisfy one ormore of the following: conjugates formed from different PEG derivatives(e.g., PEGs having different structures, reactive groups, and so forth);conjugates formed from PEG derivatives having different weight averagemolecular weights (e.g., greater than about 10,000); conjugates whereinthe polymer is not attached primarily at lysine position 52; conjugatesformed from recombinant EPO rather than muteins or fusion proteinsthereof; compositions that are substantially homogeneous in terms oftheir EPO-PEGmer content (e.g., monoPEGylated EPO, diPEGylated EPO,etc.), compositions that are well-defined and reproducible in terms ofEPO-PEGmer content and positional isoforms thereof, and conjugates thatcan be formed in relatively few steps and without the need to carry outpartial reduction steps or other synthetic transformations (e.g.,transamination reactions, addition of thiol groups, and so forth).Ideally, such a conjugate will possess suitable bioactivity in vivo, andpossess a circulating half-life such that blood or plasma levels of EPOare sustained over a longer period of time in comparison to currentlymarketed formulations—such that a pharmaceutical preparation comprisingsuch a conjugate can be administered less frequently than the currentlymarketed formulations, thereby providing a distinct advantage overcurrently available EPO formulations.

Thus, there remains a need in the art to provide additional, beneficialconjugates of water-soluble polymers and moieties having EPO activity.Among other things, one or more embodiments of the present invention istherefore directed to such conjugates as well as to compositionscomprising the conjugates and related methods as described herein, whichare believed to be new and completely unsuggested by the art.

SUMMARY OF THE INVENTION

Accordingly, a conjugate is provided, the conjugate comprising an EPOmoiety covalently attached, either directly or through a spacer moiety,to a non-peptidic water-soluble polymer. The conjugate is typicallyprovided as part of a composition comprising a plurality of conjugates,where such plurality of conjugates may include conjugates havingdifferent numbers of water-soluble polymers attached thereto (e.g., anEPO monomer having one water-soluble polymer attached thereto, an EPOdimer having two water-soluble polymers attached thereto, and so on),and/or conjugates which differ in the site or sites of attachment of thewater-soluble polymer to EPO.

In one or more embodiments of the invention a composition is provided,the composition comprising a plurality of conjugates, each conjugate inthe plurality comprised of human EPO attached, either directly orthrough a spacer moiety, to a non-peptidic water-soluble polymer,wherein the composition is not a pure composition of a single positionalisoform such as monoPEGylated EPO. That is, the composition comprises amixture of monoPEGylated EPO positional isoforms, preferably althoughnot necessarily, wherein the composition comprises (a) less than 50% ofmonoPEGylated EPO at the lysine residue at position 52, (b) less than50% of monoPEGylated EPO at the N-terminal amine, or (c) both.

In one or more embodiments of the invention a composition is provided,the composition comprising a plurality of conjugates, each conjugate inthe plurality comprised of human EPO attached, either directly orthrough a spacer moiety, to a non-peptidic water-soluble polymer,wherein the composition contains less than 50% of the conjugates havingthe non-peptidic water-soluble polymer attached to the lysine residue atposition 52 of the native human EPO.

In one or more embodiments of the invention a conjugate is provided, theconjugate comprised of an EPO moiety covalently attached, eitherdirectly or through a spacer moiety, to a non-peptidic water-solublepolymer, wherein the non-peptidic water-soluble polymer has a branchedstructure, a forked structure, or both.

In one or more embodiments of the invention a conjugate is provided, theconjugate comprising an EPO moiety covalently attached to a non-peptidicwater-soluble polymer via an amide linkage. The amide linkage is part ofa spacer moiety having an organic radical substituent (e.g., an alkylgroup) at the carbon atom alpha (α) to the carbonyl group of the amidelinkage.

According to one or more particular embodiments of the invention, aconjugate of EPO comprises the structure:

where POLY is a polyalkylene oxide, Q is an optional linking grouphaving a length of from one to 10 atoms, m is an integer ranging from 0to 20, Z is selected from the group consisting of alkyl, substitutedalkyl, aryl and substituted aryl, EPO is a residue of erythropoietin,and “—NH-EPO” represents an amino group of EPO.

Preferably, POLY is a polyethylene glycol, and may possess any of anumber of architectures, e.g., linear, branched, and/or forked.

Typically, POLY possesses a weight average molecular weight fallingwithin one of the following ranges: from about 10,000 Daltons to about100,000 Daltons, from about 10,000 Daltons to about 60,000 Daltons, fromabout 15,000 Daltons to about 50,000 Daltons, and from about 20,000Daltons to about 45,000 Daltons. In one or more particular embodiments,POLY is a PEG possessing a weight average molecular weight selected fromthe group consisting of about 20 kilodaltons, about 30 kilodaltons,about 40 kilodaltons, and about 50 kilodaltons. In one particularlypreferred embodiment, POLY is a PEG having a weight average molecularweight that is about 30 kilodaltons. In yet one or more particularembodiments, POLY is a PEG comprising an end-capping group such asmethyl, ethyl, benzyl, and the like.

In reference to the structure above, Q is an optional linking group,meaning that the group may or may not be present. In one or moreembodiments of the invention, Q is absent. In yet one or more additionalembodiments, Q is present and possesses a length ranging from one to tenatoms, preferably from one to seven atoms, or even more preferably fromone to five atoms, and may contain a heteroatom such as O, N, or S.

In reference to the structure above, m is an integer referring to thenumber of methylene subunits in the spacer moiety interposed betweenPOLY and —NH-EPO. In one or more embodiments, the integer m is selectedfrom the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.Preferably, m is an integer ranging from 1 to 6, or more preferably from1 to 5. In one or more embodiments, m is 1, 2 or 3.

In reference to the structure above, Z refers to a substituent on thecarbon alpha to the carbonyl group. In one or more embodiments, Z islower alkyl or substituted lower alkyl, e.g., methyl, ethyl, n-propyl,isopropyl, n-butyl, or isobutyl. Preferably, Z is methyl.

In one or more embodiments, a conjugate of the invention comprises thestructure:

where n ranges from about 200 to about 1400. In one or more additionalembodiments, n ranges from about 300 to about 1150, or from about 400 toabout 900.

In one or more embodiments, EPO corresponds to human EPO, e.g., SEQ IDNO:1 or SEQ ID NO:2.

In yet one or more additional embodiments, an EPO conjugate of theinvention (such as that described by the preceding structures) is amonoPEGylated EPO conjugate, that is to say, EPO having only onenon-peptidic and water-soluble polymer such as PEG covalently attachedto EPO. Also provided herein is a diPEGylated EPO conjugate, where EPOis covalently attached to only two water-soluble polymers such as PEG.The polymer may be attached to any reactive amine site on EPO, such asone or more of the following: lysine 20, 45, 52, 97, 116, 140, 153, 155,or the N-terminus. Also provided is a composition comprising bothmonoPEGylated EPO and diPEGylated EPO, e.g., corresponding to one ormore of the structures provided herein.

Also provided herein are compositions comprising a plurality of EPOconjugates. In one or more particular embodiments, a composition of theinvention is one wherein about greater than 85%, or even 90% of thePEG-EPO conjugates in the composition are mono-PEGylated. In one moreparticular embodiments, provided is a composition of monoPEGylated EPOconjugates, wherein a minority (e.g., less than 50%) of themonoPEGylated EPO conjugate species in the composition possess polymer,e.g., PEG, covalently attached at lysine 52.

In yet one or more particular embodiments, and in particular thosedescribed by the above structures, provided is a composition ofmonoPEGylated EPO conjugates, wherein a minority (e.g., less than 50%)of the monoPEGylated EPO conjugate species in the composition possess apolymer, e.g., PEG, covalently attached at the N-terminus. In apreferred embodiment, provided is a composition of monoPEGylated EPOconjugates, wherein (i) less than 50% of the monoPEGylated species inthe composition possess a polymer, e.g., PEG, covalently attached tolysine 52, and (ii) less than 50% of the monoPEGylated species in thecomposition possess a polymer, e.g., PEG, covalently attached at theN-terminus.

Also provided in one or more embodiments of the invention are EPOconjugates corresponding to one or more of the following structures:

where n corresponds to the values previously described.

Also provided is an EPO conjugate corresponding to any one or more ofthe structures provided herein, e.g., in Tables 1, 2 or 3.

In one or more embodiments of the invention, the EPO conjugate isbioactive in vivo, and possesses a bioactivity that is at least about5%, or 10%, or 15%, or 20%, or 30%, or 40% or 50% or 60% or 70% or 80%or 90% or greater that of the unmodified EPO moiety itself.

In one or more embodiments of the invention, the EPO conjugate, whenadministered in vivo, demonstrates a pharmacokinetic profile that isimproved over that of either unmodified EPO or currently marketedEPO-moieties such as Aranesp® erythropoietin.

In yet another aspect, the invention is directed, in one or moreembodiments, to a method for preparing an EPO conjugate as describedherein. Such method comprises the step of contacting one or morepolymeric reagents with an EPO moiety under conditions sufficient toresult in a conjugate comprising an EPO moiety covalently attached,either directly or through a spacer moiety comprised of one or moreatoms, to a water-soluble polymer, wherein the method for preparing theconjugate lacks a partial reduction step.

In yet another aspect, the invention is directed, in one or moreembodiments, to a method for preparing a conjugate comprising the stepof contacting one or more polymeric reagents with an EPO moiety underconditions sufficient to result in a conjugate comprising an EPO moietycovalently attached, either directly or through a spacer moietycomprised of one or more atoms, to a water-soluble polymer, wherein themethod for preparing the conjugate lacks a reduction step.

In yet another aspect, the invention is directed, in one or moreembodiments, to a method for preparing a conjugate comprising the stepof contacting one or more polymeric reagents to an EPO moiety underconditions sufficient to result in a conjugate comprising an EPO moietycovalently attached, either directly or through a spacer moietycomprised of one or more atoms, to a water-soluble polymer, wherein themethod lacks a transamination step.

In yet another aspect, the invention is directed, in one or moreembodiments, to a method for preparing a conjugate comprising the stepof contacting one or more polymeric reagents to an EPO moiety underconditions sufficient to result in a conjugate comprising an EPO moietycovalently attached, either directly or through a spacer moietycomprised of one or more atoms, to a water-soluble polymer, wherein themethod lacks a step requiring the introduction of a thiol group into theEPO moiety, or where the EPO moiety itself is not an EPO-cysteinemutein.

In yet another aspect, the invention provides a method for preparing aconjugate of EPO, whereby the method is effective to provide acomposition comprising a plurality of polyalkylene oxide conjugates ofEPO wherein a minority of the conjugate species formed possess apolyalkylene oxide covalently attached at lysine 52. The methodcomprises the steps of: (i) protecting reactive amino groups on an EPOmoiety with a cyclic dicarboxylic acid anhydride protecting reagent toform an amino-protected EPO moiety, (ii) reacting a polyalkylene oxidereagent comprising a leaving group X with the amino-protected EPO moietyunder conditions effective to promote reaction of one or moreunprotected amino sites on EPO with the polyalkylene oxide reagent, tothereby form a polyalkylene oxide-amino protected-EPO conjugate, and(iii) deprotecting the polyalkylene oxide-amino protected-EPO conjugateto provide a composition comprising a plurality of polyalkylene oxideEPO conjugates wherein a minority of the conjugate species possess apolyalkylene oxide covalently attached at lysine 52.

In yet another aspect, the invention is directed to a method forpreparing a conjugate of EPO, where the method includes the steps of:

reacting a polymer comprising the structure:

where the variables are as previously described, and X is a leavinggroup (e.g., chlorine, bromine, N-succinimidyloxy,sulfo-N-succinimidyloxy, 1-benzotriazolyloxy, hydroxyl, 1-imidazolyl,p-nitrophenyloxy, etc.,), with erythropoietin (EPO), under conditionseffective to promote reaction of one or more amino sites on EPO with thepolymer, to thereby form a biologically active conjugate comprising thestructure:

where the variables are as previously described.

In one or more embodiments of a method of preparing a polymer-EPOconjugate, the reacting step further comprises combining an aqueoussolution of the polymer reagent with an aqueous solution of EPO to forma polymer-EPO reaction mixture. The pH of the polymer-EPO reactionmixture is typically in a range or is adjusted to be in a range fromabout 5 to about 8.5, preferably from about 5 to 8, and more preferablyfrom about 5.5 to 7.5.

In yet one or more embodiments of a method for preparing a polymer-EPOconjugate, the polymer reagent is combined at a 5-fold or greater molarexcess relative to EPO, preferably at a 10-fold or greater molar excessrelative to EPO, and even more preferably at a 20-fold or greater molarexcess relative to EPO.

In one or more additional embodiments of a method of preparing apolymer-EPO conjugate, the reacting step further comprises, aftercombining, stirring the reaction mixture for about 1-24 hours at atemperature ranging from about −10° C. to about 40° C. In yet one ormore additional embodiments, stirring is carried out at ambient (e.g.,room) temperature.

In yet one or more additional embodiments of a method for preparing apolymer-EPO conjugate, the method comprises, prior to the reacting step,protecting the amino groups of EPO (typically those that are mostreactive) with a cyclic dicarboxylic acid anhydride protecting agent,e.g., a maleic or citraconylic anhydride, to form an amino-protectedEPO. Preferably, the protecting agent is dimethylmaleic anhydride.

In yet one or more additional embodiments of a method for preparing apolymer-EPO conjugate, the method further comprises, after the reactingstep, deprotecting the amino groups of the amino-protected EPO. In oneor more embodiments, such method is effective to form a conjugate of EPOwherein a minority of the conjugate species have a polyalkylene oxidecovalently attached at lysine 52, and optionally, and/or a minority ofthe conjugate species have a polyalkylene oxide covalently attached atthe N-terminus.

One or more embodiments of a method for preparing a polymer-EPOconjugate further comprise, after the reacting step, purifying theresulting conjugate(s).

In one or more embodiments of such method, the polymer reagent comprisesthe structure:

wherein X is a leaving group and n is an integer from about 200 to about1400, and the conjugate comprises the structure:

where EPO is a residue of erythropoietin and n is an integer from about200 to about 1400.

In yet one or more embodiments of the above-described method(s), thepolymer reagent comprises the structure:

wherein each n is an integer from about 200 to about 1400, and theconjugate comprises the structure:

wherein EPO is a residue of erythropoietin and each n is an integer fromabout 200 to about 1400.

In yet one or more embodiments of the above-described method(s), thepolymer reagent comprises the structure:

wherein each n is an integer from about 200 to about 1400, and theconjugate comprises the structure:

wherein each n is an integer from about 200 to about 1400 and EPO is aresidue of erythropoietin.

In one or more embodiments of the invention a composition is provided,the composition comprising a conjugate as described herein incombination with a pharmaceutically acceptable excipient. Thecompositions encompass all types of formulations and in particular thosethat are suited for injection such as powders that can be reconstituted,as well as liquids (e.g., suspensions and solutions).

In one or more embodiments of the invention, a method for administeringan EPO conjugate is provided, the method comprising the step ofadministering to a patient in need thereof a composition comprising oneor more conjugates as described herein, wherein the composition containsa therapeutically effective amount of the one or more conjugates. Thestep of administering the conjugate can be effected by injection (e.g.,intramuscular injection, intravenous injection, subcutaneous injection,and so forth). The patient may be one suffering from anemia, e.g., dueto chronic renal failure, or AIDS, or as a result of chemotherapy. Inone or more particular embodiments of a method for administering, theconjugate-comprising composition is administered once daily, every otherday, twice a week, three times a week, once a week, once every otherweek, once every three weeks, or even one a month.

Each of the herein-described features of the invention is meant to applyequally to each and every embodiment as described herein, unlessotherwise indicated.

Additional objects, advantages and novel features of the invention willbe set forth in the description that follows, and in part, will becomeapparent to those skilled in the art upon reading the following, or maybe learned by practice of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1D correspond to peptide mapping results for unmodified EPO,along with exemplary PEG-EPO conjugate samples as described in Example18. Plots correspond to total ion current based upon electrosprayionization mass spectrometric detection.

FIG. 2 is a plot demonstrating the comparative blood levels over timefor unmodified EPO, Aranesp®, and an exemplary EPO conjugate inaccordance with the invention, 01-P-MSMBE-30, when subcutaneouslyadministered to mice as described in Example 19.

DETAILED DESCRIPTION OF THE INVENTION

Before describing one or more embodiments of the present invention indetail, it is to be understood that this invention is not limited to theparticular polymers, synthetic techniques, EPO moieties, and the like,as such may vary.

It must be noted that, as used in this specification and the intendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “a polymer” includes a single polymer as well as two ormore of the same or different polymers, reference to “an optionalexcipient” refers to a single optional excipient as well as two or moreof the same or different optional excipients.

In describing and claiming one or more embodiments of the presentinvention, the following terminology will be used in accordance with thedefinitions described below.

“PEG,” “polyethylene glycol” and “poly(ethylene glycol)” as used herein,are interchangeable and meant to encompass any non-peptidicwater-soluble poly(ethylene oxide). Typically, PEGs for use inaccordance with the invention comprise the following structure“—(OCH₂CH₂)—” where (n) is 2 to 4000, preferably from 200 to 1400. Asused herein, PEG also includes “—CH₂CH₂—O(CH₂CH₂O)_(n)—CH₂CH₂—” and“—(OCH₂CH₂)_(n)O—,” depending upon whether or not the terminal oxygenshave been displaced. Throughout the specification and claims, it shouldbe remembered that the term “PEG” includes structures having variousterminal or “end capping” groups and so forth. The term “PEG” also meansa polymer that contains a majority, that is to say, greater than 50%, of—OCH₂CH₂— repeating subunits. With respect to specific forms, the PEGcan take any number of a variety of molecular weights, as well asstructures or geometries such as “branched,” “linear,” “forked,”“multifunctional,” and the like, to be described in greater detailbelow.

The terms “end-capped” and “terminally capped” are interchangeably usedherein to refer to a terminal or endpoint of a polymer having anend-capping moiety. Typically, although not necessarily, the end-cappingmoiety comprises a hydroxy or C₁₋₂₀ alkoxy group, more preferably aC₁₋₁₀ alkoxy group, and still more preferably a C₁₋₅ alkoxy group. Thus,examples of end-capping moieties include alkoxy (e.g., methoxy, ethoxyand benzyloxy), as well as aryl, heteroaryl, cyclo, heterocyclo, and thelike. It must be remembered that the end-capping moiety may include oneor more atoms of the terminal monomer in the polymer [e.g., theend-capping moiety “methoxy” in CH₃O(CH₂CH₂O)_(n)— andCH₃(OCH₂CH₂)_(n)—]. In addition, saturated, unsaturated, substituted andunsubstituted forms of each of the foregoing are envisioned. Moreover,the end-capping group can also be a silane. The end-capping group canalso advantageously comprise a detectable label. When the polymer has anend-capping group comprising a detectable label, the amount or locationof the polymer and/or the moiety (e.g., active agent) to which thepolymer is coupled can be determined by using a suitable detector. Suchlabels include, without limitation, fluorescers, chemiluminescers,moieties used in enzyme labeling, colorimetric (e.g., dyes), metal ions,radioactive moieties, and the like. Suitable detectors includephotometers, films, spectrometers, and the like. The end-capping groupcan also advantageously comprise a phospholipid. When the polymer has anend-capping group comprising a phospholipid, unique properties areimparted to the polymer and the resulting conjugate. Exemplaryphospholipids include, without limitation, those selected from the classof phospholipids called phosphatidylcholines. Specific phospholipidsinclude, without limitation, those selected from the group consisting ofdilauroylphosphatidylcholine, dioleylphosphatidylcholine,dipalmitoylphosphatidylcholine, disteroylphosphatidylcholine,behenoylphosphatidylcholine, arachidoylphosphatidylcholine, andlecithin.

“Non-naturally occurring” with respect to a polymer as described herein,means a polymer that in its entirety is not found in nature. Anon-naturally occurring polymer may, however, contain one or moremonomers or segments of monomers that are naturally occurring, so longas the overall polymer structure is not found in nature.

The term “water-soluble” as in a “water-soluble polymer” is any polymerthat is soluble in water at room temperature. Typically, a water-solublepolymer will transmit at least about 75%, more preferably at least about95%, of light transmitted by the same solution after filtering. On aweight basis, a water-soluble polymer will preferably be at least about35% (by weight) soluble in water, more preferably at least about 50% (byweight) soluble in water, still more preferably about 70% (by weight)soluble in water, and still more preferably about 85% (by weight)soluble in water. It is most preferred, however, that the water-solublepolymer is about 95% (by weight) soluble in water or completely solublein water.

Molecular weight in the context of a water-soluble polymer, such as PEG,can be expressed as either a number average molecular weight or a weightaverage molecular weight. Unless otherwise indicated, all references tomolecular weight herein refer to the weight average molecular weight.Both molecular weight determinations, number average and weight average,can be measured using gel permeation chromatography or other liquidchromatography techniques. Other methods for measuring molecular weightvalues can also be used, such as the use of end-group analysis or themeasurement of colligative properties (e.g., freezing-point depression,boiling-point elevation, or osmotic pressure) to determine numberaverage molecular weight or the use of light scattering techniques,ultracentrifugation or viscometry to determine weight average molecularweight. The polymers are typically polydisperse (i.e., number averagemolecular weight and weight average molecular weight of the polymers arenot equal), possessing low polydispersity values of preferably less thanabout 1.2, more preferably less than about 1.15, still more preferablyless than about 1.10, yet still more preferably less than about 1.05,and most preferably less than about 1.03.

The term “active” or “activated” when used in conjunction with aparticular functional group, refers to a reactive functional group thatreacts readily with an electrophile or a nucleophile on anothermolecule. This is in contrast to those groups that require strongcatalysts or highly impractical reaction conditions in order to react(i.e., a “non-reactive” or “inert” group).

As used herein, the term “functional group” or any synonym thereof ismeant to encompass protected forms thereof as well as unprotected forms.

A leaving group is an atom or molecule (charged or uncharged) that isdisplaced from a parent molecule, e.g., in a substitution or anelimination reaction.

The terms “spacer moiety,” “linkage” and “linker” are used herein torefer to an atom or a collection of atoms optionally used to linkinterconnecting moieties such as a terminus of a polymer segment and anEPO moiety or an electrophile or nucleophile of an EPO moiety. Thespacer moiety may be hydrolytically stable or may include aphysiologically hydrolyzable or enzymatically degradable linkage.Preferably, a spacer or linker in the present invention is one that ishydrolytically stable.

“Alkyl” refers to a hydrocarbon chain, typically ranging from about 1 to15 atoms in length. Such hydrocarbon chains are preferably but notnecessarily saturated and may be branched or straight chain, althoughtypically straight chain is preferred. Exemplary alkyl groups includemethyl, ethyl, propyl, butyl, pentyl, 1-methylbutyl, 1-ethylpropyl,3-methylpentyl, and the like. As used herein, “alkyl” includescycloalkyl as well as cycloalkylene-containing alkyl.

“Lower alkyl” refers to an alkyl group containing from 1 to 6 carbonatoms, and may be straight chain or branched, as exemplified by methyl,ethyl, n-butyl, i-butyl, and t-butyl.

“Cycloalkyl” refers to a saturated or unsaturated cyclic hydrocarbonchain, including bridged, fused, or spiro cyclic compounds, preferablymade up of 3 to about 12 carbon atoms, more preferably 3 to about 8carbon atoms. “Cycloalkylene” refers to a cycloalkyl group that isinserted into an alkyl chain by bonding of the chain at any two carbonsin the cyclic ring system.

“Alkoxy” refers to an —O—R group, wherein R is alkyl or substitutedalkyl, preferably C₁₋₆ alkyl (e.g., methoxy, ethoxy, propyloxy, and soforth).

The term “substituted” as in, for example, “substituted alkyl,” refersto a moiety (e.g., an alkyl group) substituted with one or morenoninterfering substituents, such as, but not limited to: alkyl, C₃₋₈cycloalkyl, e.g., cyclopropyl, cyclobutyl, and the like; halo, e.g.,fluoro, chloro, bromo, and iodo; cyano; alkoxy, lower phenyl;substituted phenyl; and the like. “Substituted aryl” is aryl having oneor more noninterfering groups as a substituent. For substitutions on aphenyl ring, the substituents may be in any orientation (i.e., ortho,meta, or para).

“Noninterfering substituents” are those groups that, when present in amolecule, are typically nonreactive with other functional groupscontained within the molecule.

“Aryl” means one or more aromatic rings, each of 5 or 6 core carbonatoms. Aryl includes multiple aryl rings that may be fused, as innaphthyl or unfused, as in biphenyl. Aryl rings may also be fused orunfused with one or more cyclic hydrocarbon, heteroaryl, or heterocyclicrings. As used herein, “aryl” includes heteroaryl.

“Heteroaryl” is an aryl group containing from one to four heteroatoms,preferably sulfur, oxygen, or nitrogen, or a combination thereof.Heteroaryl rings may also be fused with one or more cyclic hydrocarbon,heterocyclic, aryl, or heteroaryl rings.

“Heterocycle” or “heterocyclic” means one or more rings of 5-12 atoms,preferably 5-7 atoms, with or without unsaturation or aromatic characterand having at least one ring atom that is not a carbon. Preferredheteroatoms include sulfur, oxygen, and nitrogen.

“Substituted heteroaryl” is heteroaryl having one or more noninterferinggroups as substituents.

“Substituted heterocycle” is a heterocycle having one or more sidechains formed from noninterfering substituents.

“Electrophile” and “electrophilic group” refer to an ion or atom orcollection of atoms, that may be ionic, having an electrophilic center,i.e., a center that is electron seeking, capable of reacting with anucleophile.

“Nucleophile” and “nucleophilic group” refers to an ion or atom orcollection of atoms that may be ionic having a nucleophilic center,i.e., a center that is seeking an electrophilic center or with anelectrophile.

“Atom length” refers to the number of atoms making up a particularfragment, spacer, linker or the like. By atom length is meant the numberof atoms in a single chain, not counting substituents. For instance,—CH₂— counts as one atom with respect to atom length, —CH₂ CH₂ O— countsas 3 atoms in length, and so on.

A “physiologically cleavable” or “hydrolyzable” or “degradable” bond isa bond that reacts with water (i.e., is hydrolyzed) under physiologicalconditions. The tendency of a bond to hydrolyze in water will depend notonly on the general type of linkage connecting two central atoms butalso on the substituents attached to these central atoms. Appropriatehydrolytically unstable or weak linkages include but are not limited tocarboxylate ester, phosphate ester, anhydrides, acetals, ketals,acyloxyalkyl ether, imines, orthoesters, peptides and oligonucleotides.

An “enzymatically degradable linkage” means a linkage that is subject todegradation by one or more enzymes.

A “hydrolytically stable” linkage or bond refers to a chemical bond,typically a covalent bond, that is substantially stable in water, thatis to say, does not undergo hydrolysis under physiological conditions toany appreciable extent over an extended period of time. Examples ofhydrolytically stable linkages include, but are not limited to, thefollowing: carbon-carbon bonds (e.g., in aliphatic chains), ethers,amides, urethanes, and the like. Generally, a hydrolytically stablelinkage is one that exhibits a rate of hydrolysis of less than about1-2% per day under physiological conditions. Hydrolysis rates ofrepresentative chemical bonds can be found in most standard chemistrytextbooks.

“Pharmaceutically acceptable excipient or carrier” refers to anexcipient that may optionally be included in the compositions of theinvention and that causes no significant adverse toxicological effectsto the patient. “Pharmacologically effective amount,” “physiologicallyeffective amount,” and “therapeutically effective amount” are usedinterchangeably herein to mean the amount of a polymer-(EPO) moietyconjugate that is needed to provide a desired level of the conjugate (orcorresponding unconjugated EPO moiety) in the bloodstream or in thetarget tissue. The precise amount will depend upon numerous factors,e.g., the particular EPO moiety, the components and physicalcharacteristics of the therapeutic composition, intended patientpopulation, individual patient considerations, and the like, and canreadily be determined by one skilled in the art, based upon theinformation provided herein.

“Multi-functional” means a polymer having three or more functionalgroups contained therein, where the functional groups may be the same ordifferent. Multi-functional polymeric reagents of the invention willtypically contain from about 3-100 functional groups, or from 3-50functional groups, or from 3-25 functional groups, or from 3-15functional groups, or from 3 to 10 functional groups, or will contain 3,4, 5, 6, 7, 8, 9 or 10 functional groups within the polymer backbone.

The term “EPO moiety,” or simply “EPO” as used herein, refers to amoiety having EPO activity, i.e., that ability, in-vivo, to stimulatered blood cell production as well as the division and differentiation ofcommitted erythroid progenitors in the bone marrow to cause bone marrowcells to increase the production of reticulocytes and red blood cells.The EPO moiety will also have at least one electrophilic group ornucleophilic group suitable for reaction with a polymeric reagent. Inaddition, the term “EPO moiety” or “EPO” encompasses both the EPO moietyprior to conjugation as well as the EPO moiety residue followingconjugation. As will be explained in further detail below, one ofordinary skill in the art can determine whether any given moiety has EPOactivity. A protein having the amino acid sequence corresponding to SEQID NO: 1 or SEQ ID NO: 2 is an EPO moiety in accordance with theinvention, as well as any protein or polypeptide substantiallyhomologous thereto, whose biological properties result in thestimulation of red blood cell production and in the stimulation of thedivision and differentiation of committed erythroid progenitors in thebone marrow. As used herein, the term “EPO moiety” includes fragmentsand EPO modified deliberately, as for example, by site directedmutagenesis or accidentally through mutations. These terms also includeanalogs having from 1 to 6 additional glycosylation sites, analogshaving at least one additional amino acid at the carboxy terminal end ofthe protein wherein the additional amino acid(s) includes at least oneglycosylation site, and analogs having an amino acid sequence whichincludes a rearrangement of at least one glycosylation site, such as forexample the analogs disclosed in European Patent Publication No. 640619. These terms include both natural and recombinantly produced humanerythropoietin.

The term “substantially homologous” means that a particular subjectsequence, for example, a mutant sequence, varies from a referencesequence by one or more substitutions, deletions, or additions, the neteffect of which does not result in an adverse functional dissimilaritybetween the reference and subject sequences. For purposes of the presentinvention, sequences having greater than 95 percent homology, equivalentbiological properties, and equivalent expression characteristics areconsidered substantially homologous. For purposes of determininghomology, truncation of the mature sequence should be disregarded.Sequences having lesser degrees of homology, comparable bioactivity, andequivalent expression characteristics are considered substantialequivalents.

The term “fragment” of the EPO protein means any protein or polypeptidehaving the amino acid sequence of a portion or fragment of an EPOprotein, and which has the biological activity of the EPO. Fragmentsinclude proteins or polypeptides produced by proteolytic degradation ofthe EPO protein or produced by chemical synthesis by methods routine inthe art. An EPO protein or fragment thereof is biologically active whenadministration of the protein or fragment to a human results in thestimulation of red blood cell production and the stimulation of thedivision and differentiation of committed erythroid progenitors in thebone marrow. Determining such biological activity of the EPO protein cancarried out by conventional, well-known tests utilized for such purposeson one or more species of mammals. An appropriate test which can beutilized to demonstrate such biological activity is described herein.

The term “patient,” or “subject” refers to a living organism sufferingfrom or prone to a condition that can be prevented or treated byadministration of an EPO moiety (e.g., conjugate), and includes bothhumans and animals.

“Optional” or “optionally” means that the subsequently describedcircumstance may or may not occur, so that the description includesinstances where the circumstance occurs and instances where it does not.

“Substantially” means nearly totally or completely, for instance,satisfying one or more of the following: greater than 50%, 51% orgreater, 75% or greater, 80% or greater, 90% or greater, and 95% orgreater of the condition.

“Minority” as used herein, means less than 50% of a given population.For instance, a population of polymer conjugates, where a minority ofsuch conjugates have polymer attached at a given EPO amino acid site,refers to an overall population of polymer conjugates, which may or maynot be more precisely defined, where 50% or less of the definedpopulation has polymer attached at a given EPO amino acid site. Specificexemplary minority amounts include less than 50%, less than 40%, lessthan 30%, less than 25%, less than 20%, less than 15%, or even less than10% of a given population.

“Ambient” in reference to temperature, refers to the temperature of theair in a particular environment, also referred to synonymously as “roomtemperature”. Room temperature typically refers to a temperature in arange from about 16° C. to about 25° C.

Amino acid residues in peptides are abbreviated as follows:Phenylalanine is Phe or F; Leucine is Leu or L; Isoleucine is Ile or I;Methionine is Met or M; Valine is Val or V; Serine is Ser or S; Prolineis Pro or P; Threonine is Thr or T; Alanine is Ala or A; Tyrosine is Tyror Y; Histidine is His or H; Glutamine is Gln or Q; Asparagine is Asn orN; Lysine is Lys or K; Aspartic Acid is Asp or D; Glutamic Acid is Gluor E; Cysteine is Cys or C; Tryptophan is Trp or W; Arginine is Arg orR; and Glycine is Gly or G.

Turning to one or more embodiments of the invention, a conjugate isprovided, the conjugate comprising an EPO moiety covalently attached,either directly or through a spacer moiety, to a non-peptidicwater-soluble polymer. The conjugates of the invention will possess oneor more of the following features.

The EPO Moiety

As previously stated, the conjugate comprises an EPO moiety (alsoreferred to herein simply as “EPO”) covalently attached, either directlyor through a spacer moiety, to a non-peptidic water-soluble polymer. Asused herein, the term “EPO moiety” shall refer to the EPO moiety priorto conjugation as well as to the EPO moiety following attachment to anon-peptidic water-soluble polymer. In the latter instance, EPO is oftenreferred to as a residue of erythropoietin or as an EPO residue, sincethe parent EPO moiety itself is slightly altered from the unmodifiedparent, due to the covalent attachment of one or more water-solublepolymers thereto. The EPO moiety in the conjugate is any peptide thatprovides an erythropoietic effect in vivo or in vitro.

The cDNA coding for human EPO has been isolated and characterized. See,for example, U.S. Pat. No. 4,703,008. The amino acid sequence forrecombinant human EPO (“rHuEPO”) is identical to the sequence for EPOobtained from human urinary sources. The amino acid sequence for humanEPO is provided in SEQ ID NO: 1. An arginine residue-containing form ofEPO is provided in SEQ ID NO: 2. EPO can be expressed in bacterial(e.g., Escherichia coli), mammalian (e.g., Chinese hamster ovary cells),and yeast (e.g., Saccharomyces cerevisiae) expression systems, amongothers.

For any given moiety, it is possible to determine whether that moietyhas EPO activity. For example, as described in U.S. Pat. No. 5,688,679,an EPO moiety of interest can be evaluated for EPO activity by observingthe formation of erythroid colonies in a culture of mouse bone marrowcells in a plasma clot. Briefly, as described in U.S. Pat. No.5,688,679, the EPO moiety of interest is first added to the culture.After incubation for 36 to 48 hours, the plasma clots can be fixed onmicroscope slides, stained with benzidine for hemoglobin, and erythroidcolonies counted. The tested EPO moiety will have erythropoieticactivity if there are a greater number of erythroid colonies in theEPO-moiety treated culture than in a control.

Additional illustrative in vitro assays for evaluating dose-dependentproliferation activities of an EPO moiety (or an EPO moiety conjugate)include those using EPO-responsive target cells such as primary murinespleen cells (Krystal, G., 1983, Exp. Hematol. 11, 649-60), HCD 57, amurine MEL cell line (Hankins, W. D., et al., 1987, Blood, 70, 173a),and UT7-EPO, a human cell line derived from the bone marrow of a patientwith acute megakaryoblastic leukemia (Komatsu, N., et al., 1991, CancerRes. 51, 341-348).

Alternatively, EPO activity can be evaluated in vivo in rats bymonitoring changes in reticulocyte counts and hemoglobin levels after asingle tail vein injection of a particular EPO moiety (or correspondingconjugate). In vivo activity can also be measured using thenormocythaemic mouse assay (European Pharmacopoia 2002).

Nonlimiting examples of EPO moieties include the following: EPO asidentified in SEQ. ID. NO: 1 and SEQ. ID. NO: 2, as well as truncatedversions, hybrid variants, and peptide mimetics thereof. Biologicallyactive fragments, deletion variants, substitution variants or additionvariants of any of the foregoing that maintain at least some degree ofEPO activity can also serve as an EPO moiety.

Depending on the system used to express proteins having EPO activity,the EPO moiety can be unglycosylated or glycosylated and either may beused. That is, the EPO moiety can be unglycosylated or the EPO moietycan be glycosylated. In one or more embodiments of the invention, it ispreferred that the EPO moiety is not glycosylated.

The EPO moiety can advantageously be modified to include one or moreamino acid residues such as, for example, lysine, cysteine and/orarginine, in order to provide facile attachment of the polymer to anatom within the side chain of the amino acid. In addition, the EPOmoiety can be modified to include a non-naturally occurring amino acidresidue. Techniques for adding amino acid residues and non-naturallyoccurring amino acid residues are well known to those of ordinary skillin the art. Reference is made to the following: Roe, B., et al., Ed.,“Protocols for Recombinant DNA Isolation, Cloning and Sequencing”, 1996,Wiley & Sons.; Ausubel, F. M., Ed., “Short Protocols in MolecularBiology”, 5^(th) Ed., Wiley & Sons; J. March, “Advanced OrganicChemistry: Reactions Mechanisms and Structure”, 4th Ed. (New York:Wiley-Interscience, 1992); Wen, D., et al., J. Biol. Chem., 1994, 269(36), 22839. In one or more embodiments of the invention, it ispreferred that the EPO moiety is not modified to include one or moreamino acid residues.

In addition, the EPO moiety can advantageously be modified to includeattachment of a functional group (other than through addition of afunctional group-containing amino acid residue). In addition, the EPOmoiety can be modified to include an N-terminal alpha carbon. Inaddition, the EPO moiety can be modified to include one or morecarbohydrate moieties. In some embodiments of the invention, it ispreferred that the EPO moiety is not modified to include a thiol groupand/or an N-terminal alpha carbon.

The EPO moiety can be obtained from any conventional source such astissues, protein synthesis, or cell culture with natural or recombinantcells. Preferred are recombinant methods. Briefly, recombinant methodsinvolve constructing the nucleic acid encoding the desired polypeptideor fragment, cloning the nucleic acid into an expression vector,transforming a host cell (e.g., plant, bacteria, yeast, transgenicanimal cell, or mammalian cell such as Chinese hamster ovary cell orbaby hamster kidney cell), and expressing the nucleic acid to producethe desired polypeptide or fragment. Methods for producing andexpressing recombinant polypeptides in vitro and in prokaryotic andeukaryotic host cells are known to those of ordinary skill in the art,and are disclosed, for example, in U.S. Pat. Nos. 5,733,761, 5,641,670,and 5,733,746, among others.

To facilitate identification and purification of the recombinantpolypeptide, nucleic acid sequences that encode for an epitope tag orother affinity binding sequence can be inserted or added in-frame withthe coding sequence, thereby producing a fusion protein comprised of thedesired polypeptide and a polypeptide suited for binding. Fusionproteins can be identified and purified by first running a mixturecontaining the fusion protein through an affinity column bearing bindingmoieties (e.g., antibodies) directed against the epitope tag or otherbinding sequence in the fusion proteins, thereby binding the fusionprotein within the column. Thereafter, the fusion protein can berecovered by washing the column with the appropriate solution (e.g.,acid) to release the bound fusion protein. The recombinant polypeptidecan also be identified and purified by lysing the host cells, separatingthe polypeptide, e.g., by size exclusion chromatography, and collectingthe polypeptide. These and other methods for identifying and purifyingrecombinant polypeptides are known to those of ordinary skill in theart. In one or more embodiments of the invention, however, it ispreferred that the EPO moiety is not in the form of a fusion protein.

A preferred EPO moiety has the amino acid sequence as provided in SEQ IDNO: 1. Unless specifically noted, all assignments of a numeric locationof an amino acid residue as provided herein are based on SEQ ID NO: 1.An additionally described amino acid sequence that corresponds toanother EPO moiety is provided in SEQ ID NO: 2. Commercially availableversions of EPO moieties are available such as PROCLAT® EPO (DragonPharmaceuticals, Inc., Vancouver, B.C., Canada), EPDX EPO (Mumbai,India), human cell-expressed recombinant human EPO (Apollo CytokineResearch), ARANESP®, and PROCRIT® EPO (Ortho Biotech Products, L.P.,(Bridgewater, N.J.).

The Non-Peptidic Water-Soluble Polymer

As previously discussed, each conjugate comprises an EPO moiety attachedto a non-peptidic water-soluble polymer. With respect to thenon-peptidic water-soluble polymer, the non-peptidic water-solublepolymer is non-peptidic, nontoxic, non-naturally occurring andbiocompatible. With respect to biocompatibility, a substance isconsidered biocompatible if the beneficial effects associated with useof the substance alone or with another substance (e.g., an active agentsuch as an EPO moiety) in connection with living tissues (e.g.,administration to a patient) outweighs any deleterious effects asevaluated by a clinician, e.g., a physician. With respect tonon-immunogenicity, a substance is considered nonimmunogenic if theintended use of the substance in vivo does not produce an undesiredimmune response (e.g., the formation of antibodies) or, if an immuneresponse is produced, that such a response is not deemed clinicallysignificant or important as evaluated by a clinician. It is particularlypreferred that the non-peptidic water-soluble polymer is biocompatibleand nonimmunogenic.

Further, the polymer is typically characterized as having from 2 toabout 300 termini. Examples of such polymers include, but are notlimited to, poly(alkylene glycols) such as polyethylene glycol (PEG),poly(propylene glycol) (“PPG”), copolymers of ethylene glycol andpropylene glycol and the like, poly(oxyethylated polyol), poly(olefinicalcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide),poly(hydroxyalkylmethacrylate), poly(saccharides), poly(α-hydroxy acid),poly(vinyl alcohol), polyphosphazene, polyoxazoline,poly(N-acryloylmorpholine), and combinations of any of the foregoing.

The polymer is not limited to a particular structure and can be linear(e.g., alkoxy PEG or bifunctional PEG), branched or multi-armed (e.g.,forked PEG or PEG attached to a polyol core), dendritic, or withdegradable linkages. Moreover, the internal structure of the polymer canbe organized in any number of different patterns and can be selectedfrom the group consisting of homopolymer, alternating copolymer, randomcopolymer, block copolymer, alternating tripolymer, random tripolymer,and block tripolymer.

Typically, activated PEG and other activated water-soluble polymers(i.e., polymeric reagents) are activated with a suitable activatinggroup appropriate for coupling to a desired site on the EPO moiety.Thus, a polymeric reagent will possess a reactive group for reactionwith the EPO moiety. Representative polymeric reagents and methods forconjugating these polymers to an active moiety are known in the art andfurther described in Zalipsky, S., et al., “Use of FunctionalizedPoly(Ethylene Glycols) for Modification of Polypeptides” in PolyethyleneGlycol Chemistry: Biotechnical and Biomedical Applications, J. M.Harris, Plenus Press, New York (1992), and in Zalipsky (1995) AdvancedDrug Reviews 16:157-182.

Typically, the weight-average molecular weight of the non-peptidicwater-soluble polymer in the conjugate is from about 100 Daltons toabout 150,000 Daltons. Exemplary ranges, however, include weight-averagemolecular weights in the range of greater than 5,000 Daltons to about100,000 Daltons, in the range of from about 6,000 Daltons to about90,000 Daltons, in the range of from about 10,000 Daltons to about85,000 Daltons, in the range of greater than 10,000 Daltons to about60,000 Daltons, in the range of from about 20,000 Daltons to about85,000 Daltons, in the range of from about 53,000 Daltons to about85,000 Daltons, in the range of from about 25,000 Daltons to about120,000 Daltons, in the range of from about 29,000 Daltons to about120,000 Daltons, in the range of from about 35,000 Daltons to about120,000 Daltons, and in the range of from about 40,000 Daltons to about120,000 Daltons. For any given non-peptidic water-soluble polymer, PEGshaving a molecular weight in one or more of these ranges are preferred.

Exemplary weight-average molecular weights for the non-peptidicwater-soluble polymer include about 100 Daltons, about 200 Daltons,about 300 Daltons, about 400 Daltons, about 500 Daltons, about 600Daltons, about 700 Daltons, about 750 Daltons, about 800 Daltons, about900 Daltons, about 1,000 Daltons, about 1,500 Daltons, about 2,000Daltons, about 2,200 Daltons, about 2,500 Daltons, about 3,000 Daltons,about 4,000 Daltons, about 4,400 Daltons, about 5,000 Daltons, about5,500 Daltons, about 6,000 Daltons, about 7,000 Daltons, about 7,500Daltons, about 8,000 Daltons, about 9,000 Daltons, about 10,000 Daltons,about 11,000 Daltons, about 12,000 Daltons, about 13,000 Daltons, about14,000 Daltons, about 15,000 Daltons, about 20,000 Daltons, about 22,500Daltons, about 25,000 Daltons, about 30,000 Daltons, about 35,000Daltons, about 40,000 Daltons, about 45,000 Daltons, about 50,000Daltons, about 55,000 Daltons, about 60,000 Daltons, about 65,000Daltons, about 70,000 Daltons, and about 75,000 Daltons. Branchedversions of the water-soluble polymer (e.g., a branched 40,000 Daltonwater-soluble polymer comprised of two 20,000 Dalton polymers) having atotal molecular weight of any of the foregoing can also be used. In oneor more embodiments, the conjugate will not have any PEG moietiesattached, either directly or indirectly, with a PEG having a weightaverage molecular weight of less than about 6,000 Daltons.

When used as the polymer, PEG will typically comprise a number of(—OCH₂CH₂—) monomers [or (—CH₂CH₂O—) monomers, depending on how the PEGis defined]. As used throughout the description, the number of repeatingunits is identified by the subscript “n” in “(OCH₂CH₂)_(n).” Thus, thevalue of (n) typically falls within one or more of the following ranges:from 2 to about 3400, from about 100 to about 2300, from about 100 toabout 2270, from about 136 to about 2050, from about 200 to about 1400,from about 225 to about 1930, from about 450 to about 1930, from about1200 to about 1930, from about 568 to about 2727, from about 660 toabout 2730, from about 795 to about 2730, from about 795 to about 2730,from about 909 to about 2730, and from about 1,200 to about 1,900. Forany given polymer in which the molecular weight is known, it is possibleto determine the number of repeating units (i.e., “n”) by dividing thetotal weight-average molecular weight of the polymer by the molecularweight of the repeating monomer.

One particularly preferred polymer for use in the invention is anend-capped polymer, that is, a polymer having at least one terminuscapped with a relatively inert group, such as a lower C₁₋₆ alkoxy group,although a hydroxyl group can also be used. When the polymer is PEG, forexample, it is preferred to use a methoxy-PEG (commonly referred to asmPEG), which is a linear form of PEG wherein one terminus of the polymeris a methoxy (—OCH₃) group, while the other terminus is a hydroxyl orother functional group that can be optionally chemically modified.

In one form useful in one or more embodiments of the present invention,free or unbound PEG is a linear polymer terminated at each end withhydroxyl groups:

HO—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—OH,

wherein (n) typically ranges from zero to about 4,000.

The above polymer, alpha-, omega-dihydroxylpoly(ethylene glycol), can berepresented in brief form as HO-PEG-OH where it is understood that the—PEG- symbol can represent the following structural unit:

—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—,

wherein (n) is as defined as above.

Another type of PEG useful in one or more embodiments of the presentinvention is methoxy-PEG-OH, or mPEG in brief, in which one terminus isthe relatively inert methoxy group, while the other terminus is ahydroxyl group. The structure of mPEG is given below.

CH₃O—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—OH

wherein (n) is as described above.

Multi-armed or branched PEG molecules, such as those described in U.S.Pat. No. 5,932,462, can also be used as the PEG polymer. For example,PEG can comprise the structure:

wherein:

poly_(a) and poly_(b) are PEG backbones (either the same or different),such as methoxy poly(ethylene glycol);

R″ is a nonreactive moiety, such as H, methyl or a PEG backbone; and

P and Q are nonreactive linkages. In a preferred embodiment, thebranched PEG polymer is methoxy poly(ethylene glycol) disubstitutedlysine. Depending on the specific EPO moiety used, the reactive esterfunctional group of the disubstituted lysine may be further modified toform a functional group suitable for reaction with the target groupwithin the EPO moiety.

In addition, the PEG can comprise a forked PEG. An example of a forkedPEG is represented by the following structure:

wherein: X is a spacer moiety of one or more atoms and each Z is anactivated terminal group linked to CH by a chain of atoms of definedlength. International Patent Application No. PCT/US99/05333 disclosesvarious forked PEG structures capable of use in one or more embodimentsof the present invention. The chain of atoms linking the Z functionalgroups to the branching carbon atom serve as a tethering group and maycomprise, for example, alkyl chains, ether chains, ester chains, amidechains and combinations thereof.

The PEG polymer may comprise a pendant PEG molecule having reactivegroups, such as carboxyl, covalently attached along the length of thePEG rather than at the end of the PEG chain. The pendant reactive groupscan be attached to the PEG directly or through a spacer moiety, such asan alkylene group.

In addition to the above-described forms of PEG, the polymer can also beprepared with one or more weak or degradable linkages in the polymer,including any of the above-described polymers. For example, PEG can beprepared with ester linkages in the polymer that are subject tohydrolysis. As shown below, this hydrolysis results in cleavage of thepolymer into fragments of lower molecular weight:

-PEG-CO₂-PEG-+H₂O→-PEG-CO₂H+HO-PEG-

Other hydrolytically degradable linkages, useful as a degradable linkagewithin a polymer backbone, include: carbonate linkages; imine linkagesresulting, for example, from reaction of an amine and an aldehyde (see,e.g., Ouchi et al. (1997) Polymer Preprints 38(1):582-3); phosphateester linkages formed, for example, by reacting an alcohol with aphosphate group; hydrazone linkages which are typically formed byreaction of a hydrazide and an aldehyde; acetal linkages that aretypically formed by reaction between an aldehyde and an alcohol;orthoester linkages that are, for example, formed by reaction between aformate and an alcohol; amide linkages formed by an amine group, e.g.,at an end of a polymer such as PEG, a carboxyl group of another PEGchain; urethane linkages formed from reaction of, e.g., a PEG with aterminal isocyanate group and a PEG alcohol; peptide linkages formed byan amine group, e.g., at an end of a polymer such as PEG, and a carboxylgroup of a peptide; and oligonucleotide linkages formed by, for example,a phosphoramidite group, e.g., at the end of a polymer, and a 5′hydroxyl group of an oligonucleotide.

Such optional features of the conjugate, i.e., the introduction of oneor more degradable linkages into the polymer chain, may provide foradditional control over the final desired pharmacological properties ofthe conjugate upon administration. For example, a large and relativelyinert conjugate (i.e., having one or more high molecular weight PEGchains attached thereto, for example, one or more PEG chains having amolecular weight greater than about 10,000, wherein the conjugatepossesses essentially no bioactivity) may be administered, which is thenhydrolyzed in vivo to generate a bioactive conjugate possessing aportion of the original PEG chain. In this way, the properties of theconjugate can be more effectively tailored to balance the bioactivityand circulating half-life of the conjugate over time.

The water-soluble polymer associated with the conjugate can also be“cleavable.” That is, the water-soluble polymer cleaves (either throughhydrolysis, enzymatic processes, or otherwise), thereby resulting in theunconjugated EPO moiety. In some instances, cleavable polymers detachfrom the EPO moiety in vivo without leaving any fragment of thewater-soluble polymer. In other instances, cleavable polymers detachfrom the EPO moiety in vivo leaving a relatively small fragment (e.g., asuccinate tag) from the water-soluble polymer. An exemplary cleavablepolymer includes one that attaches to the EPO moiety via a carbonatelinkage.

Those of ordinary skill in the art will recognize that the foregoingdiscussion concerning non-peptidic and water-soluble polymers by nomeans exhaustive and is merely illustrative, and that all polymericmaterials having the qualities described above are contemplated. As usedherein, the term “polymeric reagent” generally refers to an entiremolecule, which can comprise a water-soluble polymer segment, anoptional spacer or linker moiety, and a functional group.

As described above, a conjugate of the invention comprises awater-soluble polymer covalently attached to an EPO moiety. Typically,for any given conjugate, there will be one to three water-solublepolymers covalently attached to one or more moieties having EPOactivity. In some instances, however, the conjugate may have 1, 2, 3, 4,5, 6, 7, 8 or more water-soluble polymers individually attached to anEPO moiety.

The particular linkage within the moiety having EPO activity and thepolymer depends on a number of factors. Such factors include, forexample, the particular linkage chemistry employed, the particular EPOmoiety, the available functional groups within the EPO moiety (eitherfor attachment to a polymer or conversion to a suitable attachmentsite), the presence of additional reactive functional groups within theEPO moiety, and the like.

The conjugates of the invention can be, although not necessarily,prodrugs, meaning that the linkage between the polymer and the EPOmoiety is hydrolytically degradable to allow release of the parentmoiety. Exemplary degradable linkages include carboxylate ester,phosphate ester, thiolester, anhydrides, acetals, ketals, acyloxyalkylether, imines, orthoesters, peptides and oligonucleotides. Such linkagescan be readily prepared by appropriate modification of either the EPOmoiety (e.g., the carboxyl group C terminus of the protein or a sidechain hydroxyl group of an amino acid such as serine or threoninecontained within the protein) and/or the polymeric reagent usingcoupling methods commonly employed in the art. Most preferred, however,are hydrolyzable linkages that are readily formed by reaction of asuitably activated polymer with a non-modified functional groupcontained within the moiety having EPO activity.

Alternatively, a hydrolytically stable linkage, such as an amide,urethane (also known as carbamate), amine, thioether (also known assulfide), or urea (also known as carbamide) linkage can also be employedas the linkage for coupling the EPO moiety. Again, a preferredhydrolytically stable linkage is an amide. In one approach, awater-soluble polymer bearing an activated ester can be reacted with anamine group on the EPO moiety to thereby result in an amide linkage.

The conjugates (as opposed to an unconjugated EPO moiety) may or may notpossess a measurable degree of EPO activity. That is to say, apolymer-EPO moiety conjugate in accordance with the invention willpossesses anywhere from about 0.1% to about 100% of the bioactivity ofthe unmodified parent EPO moiety. In some instances, the polymer-EPOmoiety conjugates may posses greater than 100% bioactivity of theunmodified parent EPO moiety. Preferably, compounds possessing little orno EPO activity typically contain a hydrolyzable linkage connecting thepolymer to the moiety, so that regardless of the lack of activity in theconjugate, the active parent molecule (or a derivative thereof) isreleased upon aqueous-induced cleavage of the hydrolyzable linkage. Suchactivity may be determined using a suitable in vivo or in vitro model,depending upon the known activity of the particular moiety having EPOactivity employed.

For conjugates possessing a hydrolytically stable linkage that couplesthe moiety having EPO activity to the polymer, the conjugate willtypically possess a measurable degree of bioactivity. For instance, suchconjugates are typically characterized as having a bioactivity of atleast about 2%, 5%, 10%, 15%, 25%, 30%, 40%, 50%, 60%, 80%, 85%, 90%,95% 97%, 100%, or more relative to that of the unconjugated EPO moiety,when measured in a suitable model, such as those well known in the art.Preferably, conjugates having a hydrolytically stable linkage (e.g., anamide linkage) will possess at least some degree of the bioactivity ofthe unmodified parent moiety having EPO activity.

Exemplary conjugates in accordance with the invention will now bedescribed wherein the EPO moiety is a protein. Typically, such a proteinis expected to share (at least in part) a similar amino acid sequence ashuman EPO. Thus, while reference will be made to specific locations oratoms within the native human EPO protein, such a reference is forconvenience only and one having ordinary skill in the art will be ableto readily determine the corresponding location or atom in othermoieties having EPO activity. In particular, the description providedherein for native human EPO is often applicable to fragments, deletionvariants, substitution variants or addition variants of any of theforegoing.

Amino groups on EPO moieties provide a point of attachment between theEPO moiety and the water-soluble polymer. In one embodiment, theconjugate has one water-soluble conjugate attached at the N-terminal ofthe EPO moiety, in some instances, however, the composition will containless than 50% of monoPEGylated conjugates having covalent attachment ofthe PEG moiety at the N-terminus. Human EPO comprises eightamine-containing lysine residues and one amino terminus (see SEQ ID NO:1). Thus, exemplary attachment points of this EPO include attachment atthe amine-containing side chain associated with a lysine at any one ofpositions 20, 45, 52, 97, 116, 140, 152 and 154. In some embodiments ofthe invention, it is preferred that attachment to lysine does not occurat lysine in position 52. In this embodiment, the composition willideally contain less than 50% of conjugates having attachment at thelysine-52 position. In yet another embodiment, the composition may evenbe substantially free of such conjugates.

While not wishing to be bound by theory, it is believed that theattachment of a polymer at the lysine-52 position results in a conjugatehaving reduced or compromised activity. Exemplary attachment pointsother than the amine-containing side chain associated with lysine-52include attachment at the amine-containing side chain associated with alysine at any one of positions 20, 45, 97, 116, 140, 152 and 154.Consequently, these EPO moieties (as well as most any peptidic EPOmoiety) have several amines available for participation in conjugatingreactions. Covalent attachment of a polymer reagent at lysine 52 can besubstantially avoided by the use of a reversible protecting agent suchas a cyclic dicarboxylic anhydride. The protecting reagent partiallyprotects the most reactive amino groups in the EPO moiety, such aslysine 52, thereby modifying the profile of conjugates typically formedin a random PEGylation approach.

There are a number of examples of suitable polymeric reagents useful forforming covalent linkages with available amines of an EPO moiety.Specific examples, along with the corresponding conjugate, are providedin Table 1 below. In the table, the variable (n) represents the numberof repeating monomeric units (as previously described) and “—NH-(EPO)”or “NH-EPO” represents the residue of the EPO moiety followingconjugation to the polymeric reagent. While each polymeric portion[e.g., (OCH₂CH₂)_(n) or (CH₂CH₂O)_(n)] presented in Table 1 terminatesin a “CH₃” group, other groups (such as H and benzyl) can be substitutedtherefor.

TABLE 1 Amine-Specific Polymeric Reagents and the EPO Moiety ConjugateFormed Therefrom Polymeric Reagent Corresponding Conjugate

mPEG-Succinimidyl Reagent

Amide Linkage

mPEG-Oxycarbonylimidazole Reagent

Carbamate Linkage

mPEG Nitrophenyl Reagent

Carbamate Linkage

mPEG-Trichlorophenyl Carbonate Reagent

Carbamate Linkage

mPEG-Succinimidyl Reagent

Amide Linkage

Homobifunctional PEG-Succinimidyl Reagent

Amide Linkages

mPEG-Succinimdyl Reagent

Amide Linkage

mPEG Succinimidyl Reagent

Amide Linkage

mPEG-Benzotriazole Carbonate Reagent

Carbamate Linkage

mPEG-Succinimidyl Reagent

Carbamate Linkage

mPEG-Succinimidyl Reagent

Amide Linkage

mPEG Succinimidyl Reagent

Amide Linkage

Branched mPEG2-N-Hydroxysuccinimide Reagent

Amide Linkage

mPEG-Succinimidyl Reagent

Amide Linkage

mPEG-Succinimidyl Reagent

Amide Linkage

Homobifunctianal PEG-Succinimidyl Reagent

Amide Linkages

mPEG-Succinimidyl Reagent

Amide Linkage

Homobifunctional PEG-Succinimidyl Propionate Reagent

Amide Linkages

mPEG-Succinimidyl Reagent

Amide Linkage

Branched mPEG2-N-Hydroxysuccinimide Reagent

Amide Linkage

Branched mPEG2-N-Hydroxysuccinimide Reagent

Amide Linkage

mPEG-Thioester Reagent

Amide Linkage (typically to EPO moiety having an N-terminal cysteine orhistidine)

Homobifunctional PEG Propionaldehyde Reagent

Secondary Amine Linkages

mPEG Propionaldehyde Reagent

Secondary Amine Linkage

Homobifunctional PEG Butyraldehyde Reagent

Secondary Amine Linkage

mPEG Butryaldehyde Reagent

Secondary Amine Linkage

mPEG Butryaldehyde Reagent

Secondary Amine Linkage

Homobifunctional PEG Butryaldehyde Reagent

Secondary Amine Linkages

Branched mPEG2 Butyraldehyde Reagent

Secondary Amine Linkage

Branched mPEG2 Butyraldehyde Reagent

Secondary Amine Linkage

mPEG Acetal Reagent

Secondary Amine Linkage

mPEG Piperidone Reagent

Secondary Amine Linkage (to a secondary carbon)

mPEG Methylketone Reagent

secondary amine linkage (to a secondary carbon)

mPEG Tresylate Reagent

Secondary Amine Linkage

mPEG Maleimide Reagent (under certain reaction conditions such as pH >8)

Secondary Amine Linkage

mPEG Maleimide Reagent (under certain reaction conditions such as pH >8)

Secondary Amine Linkage

mPEG Maleimide Reagent (under certain reaction conditions such as ph >8)

Secondary Amine Linkage

mPEG Forked Maleimide Reagent (under certain reaction conditions such aspH > 8)

Secondary Amine Linkages

Branched mPEG2 Maleimide Reagent (under certain reaction conditions suchas pH > 8)

Secondary Amine Linkage

Heterobifunctional PEG-Succinimidyl Reagent

Amide Linkage

Branched mPEG2-Aldehyde Reagent

Secondary Amine Linkage

Conjugation of a polymeric reagent to an amino group of an EPO moietycan be accomplished by one of ordinary skill in the art without undueexperimentation. In one approach, an EPO moiety is conjugated to anactivated polymeric reagent comprising at least one leaving group, e.g.,a succinimidyl derivative such as N-hydroxysuccinimide (NHS) or anyother suitable leaving group. For example, the polymer reagent bearingan NHS or other leaving group is reacted with the EPO moiety, typicallyin aqueous media, at a pH of about 5.5 to 8.5. The reaction is typicallycarried out in a non-amine containing buffer such as phosphate buffer(e.g., sodium or potassium phosphate), HEPES, MES, PBS, MES, or sodiumacetate. Reaction times will vary depending upon reaction temperature,although reactions are typically complete in from about 0.5 hours toabout 48 hours. Typically, the coupling reaction is carried out attemperatures ranging from about −10° C. to about 40° C., and result inthe covalent attachment of the polymer to the EPO moiety.

Depending upon the particular reactivity of the polymeric reagentemployed, the polymeric reagent is combined with EPO at one of thefollowing stoichiometries: 0.5:1 molar ratio of polymeric reagent to EPOmoiety per se or greater, 1:1 or greater, 2:1 or greater, 3:1 orgreater, 4:1 or greater, 5:1 or greater, 10:1 or greater, 15:1 orgreater, 20:1 or greater, and 25:1 or greater.

As described above, in one or more embodiments of the method, theconjugation reaction is modified by using a pH-reversibleamino-protective agent such as a cyclic dicarboxylic acid anhydride. Inthe method, prior to reaction with an activated polymeric reagent, theEPO moiety is mixed with the amino-protective agent, e.g., a maleic orcitraconylic anhydride, which partially reacts with the most reactiveamino sites in EPO to form an amino-protected EPO moiety. Thisamino-protected EPO moiety is then reacted with the activated polymericreagent under conditions effective to provide a polymer-amino-protectedEPO moiety conjugate, which is then deprotected (e.g., by altering thepH) to provide the desired polymer-EPO moiety conjugate, preferablywherein the resulting EPO-conjugate composition contains a minority ofconjugate species having polymer covalently attached at lysine 52. Evenmore preferably, in one or more embodiments, the resulting conjugatecomposition additionally contains a minority of conjugate species havingpolymer covalently attached to the N-terminus of the EPO-moiety. Onepreferred maleic anhydride is dimethylmaleic anhydride (Tsunoda, S., etal., J of Pharmacology and Experimental Therapeutics, 1999, 290 (1),368-372).

In addition, an amide linkage can similarly be formed by reacting anamine-terminated non-peptidic water-soluble polymer with an EPO moietybearing an activating a carboxylic acid group.

Typical of another approach useful for conjugating the EPO moiety to apolymeric reagent is reductive amination to conjugate a primary amine ofan EPO moiety to a polymeric reagent functionalized with a ketone,aldehyde or hydrated forms thereof (e.g., ketone hydrate, aldehydehydrate). In this approach, the primary amine from the EPO moiety reactswith the carbonyl group of the aldehyde or ketone (or the correspondinghydroxyl-containing group of a hydrated aldehyde or ketone), therebyforming a Schiff base. The Schiff base, in turn, is then reduced to astable conjugate through use of a reducing agent such as sodiumborohydride or sodium cyanoborohydride. Selective reactions (e.g., atthe N-terminus) are possible, particularly with a polymer functionalizedwith a ketone or an alpha-methyl branched aldehyde and/or under specificreaction conditions (e.g., reduced pH).

Preferred amine groups in EPO that can serve as a site for attaching apolymer include those amine groups found within a lysine residue. Inaddition, the N-terminus of any EPO moiety that is a protein can serveas a polymeric attachment site.

Carboxyl groups represent another functional group that can serve as apoint of attachment on the EPO moiety. Structurally, the conjugate willcomprise the following:

where (EPO) and the adjacent carbonyl group corresponds to thecarboxyl-containing EPO moiety residue, X is a linkage, preferably aheteroatom selected from O, —NH, and S, and POLY is a water-solublepolymer such as PEG, optionally terminating in an end-capping moiety.

The —C(O)—X linkage results from the reaction between a polymericderivative bearing a terminal functional group and a carboxyl-containingEPO moiety. As discussed above, the specific linkage will depend on thetype of functional group utilized. If the polymer is end-functionalizedor “activated” with a hydroxyl group, or an activated ester or the like,the resulting linkage will be a carboxylic acid ester and X will be O.If the polymer backbone is functionalized with a thiol group, theresulting linkage will be a thioester and X will be S. When certainmulti-arm, branched or forked polymers are employed, the C(O)X moiety,and in particular the X moiety, may be relatively more complex and mayinclude a longer linkage structure.

Water-soluble derivatives containing a hydrazide moiety are also usefulfor conjugation at carboxyl groups as illustrated by the reagents andcorresponding conjugates in the following table. In the tables herein,“(EPO)” and “EPO” are used interchangeably and refer to the EPO moietyfollowing conjugation.

TABLE 2 Carboxyl-Specific Polymeric Reagents and the EPO MoietyConjugate Formed Therefrom Polymeric Reagent Corresponding Conjugate

mPEG-Hydrazine Reagent

Hydrazone Linkage

mPEG-Hydrazine Reagent

Hydrazone Linkage

mPEG-Hydrazine Reagent

Hydrazone Linkage

mPEG-Hydrazine Reagent

Hydrazone Linkage

mPEG-Hydrazine Reagent

Hydrazone Linkage

mPEG-Hydrazine Reagent

Hydrazone Linkage

mPEG-Hydrazine Reagent

Hydrazone Linkage

mPEG-Hydrazine Reagent

Hydrazone Linkage

Thiol groups contained within the EPO moiety can serve as effectivesites of attachment for the water-soluble polymer. In particular,cysteine residues provide thiol groups when the EPO moiety is a protein.The thiol groups in such cysteine residues can then reacted with anactivated PEG that is specific for reaction with thiol groups, e.g., anN-maleimidyl polymer or other derivative, as described in U.S. Pat. No.5,739,208 and in International Patent Publication No. WO 01/62827.

With respect to both SEQ ID NO: 1 and SEQ ID NO: 2, there are fourthiol-containing cysteine residues. Thus, preferred thiol attachmentsites are associated with cysteine residues at any position 7, 29, 33,and 161. To the extent that each of the cysteine residues within EPOparticipate in disulfide bonding, it is preferred not to disrupt suchbonds as disruption of the tertiary structure of the EPO moiety mightoccur and result in potentially significantly decreased EPO activity.Thus, to the extent that any particular EPO moiety lacks a thiol groupor disruption of disulfide bonds is to be avoided, it is possible to adda cysteine residue to the EPO moiety using conventional synthetictechniques. See, for example, WO 90/12874. In addition, conventionalgenetic engineering processes can also be used to introduce a cysteineresidue into the EPO moiety. In some embodiments, however, it ispreferred not to introduce and additional cysteine residue and/or thiolgroup.

Specific examples, along with the corresponding conjugate, are providedin Table 3, below. In the table, the variable (n) represents the numberof repeating monomeric units and “—S-(EPO)” represents the EPO moietyresidue following conjugation to the water-soluble polymer. While eachpolymeric portion [e.g., (OCH₂CH₂)_(n) or (CH₂CH₂O)_(n)] presented inTable 3 terminates in a “CH₃” group, other groups (such as H and benzyl)can be substituted therefor.

TABLE 3 Thiol-Specific Polymeric Reagents and the EPO Moiety ConjugateFormed Therefrom Polymeric Reagent Corresponding Conjugate

mPEG Maleimide Reagent

Thioether Linkage

mPEG Maleimide Reagent

Thioether Linkage

mPEG Maleimide Reagent

Thioether Linkage

Homobifunctional mPEG Maleimide Reagent

Thioether Linkages

mPEG Maleimide Reagent

Thioether Linkage

mPEG Maleimide Reagent

Thioether Linkage

mPEG Forked Maleimide Reagent

Thioether Linkage

Branched mPEG2 Maleimide Reagent

Thioether Linkage

Branched mPEG2 Maleimide Reagent

Thioether Linkage

Branched mPEG2 Forked Maleimide Reagent

Thioether Linkages

Branched mPEG2 Forked Maleimide Reagent

Thioether Linkages

mPEG Vinyl Sulfone Reagent

Thioether Linkage

mPEG Thiol Reagent

Disulfide Linkage

Homobifunctional PEG Thiol Reagent

Disulfide Linkages

mPEG Disulfide Reagent

Disulfide Linkage

Homobifunctional Disulfide Reagent

Disulfide Linkages

With respect to conjugates formed from water-soluble polymers bearingone or more maleimide functional groups (regardless of whether themaleimide reacts with an amine or thiol group on the EPO moiety), thecorresponding maleamic acid form(s) of the water-soluble polymer canalso react with the EPO moiety. Under certain conditions (e.g., a pH ofabout 7-9 and in the presence of water), the maleimide ring will “open”to form the corresponding maleamic acid. The maleamic acid, in turn, canreact with an amine or thiol group of an EPO moiety. Exemplary maleamicacid-based reactions are schematically shown below. POLY represents thewater-soluble polymer, and (EPO) represents the EPO moiety.

A representative conjugate in accordance with the invention can have thefollowing structure:

POLY-L_(0,1)-C(O)Z—Y—S—S-(EPO)

wherein POLY is a water-soluble polymer, L is an optional linker, Z is aheteroatom selected from the group consisting of O, NH, and S, and Y isselected from the group consisting of C₂₋₁₀ alkyl, C₂₋₁₀ substitutedalkyl, aryl, and substituted aryl, and (EPO) is an EPO moiety. Polymericreagents that can be reacted with an EPO moiety and result in this typeof conjugate are described in U.S. Patent Application Publication No.2005/0014903.

Preferred thiol groups in an EPO moiety that can serve as a site forattaching a polymeric reagent include those thiol groups found withincysteine residues.

With respect to polymeric reagents, those described here and elsewherecan be purchased from commercial sources (e.g., Nektar Therapeutics,Huntsville Ala.). In addition, methods for preparing the polymericreagents are described in the literature.

The attachment between the EPO moiety and the non-peptidic water-solublepolymer can be direct, wherein no intervening atoms are located betweenthe EPO moiety and the polymer, or indirect, wherein one or more atomsare located between the EPO moiety and the polymer. With respect to theindirect attachment, the one or more atoms is conventionally referred toas a “spacer moiety,” or as a “linking moiety or linking group” whichcan include one or more of carbon atoms, nitrogen atoms, sulfur atoms,oxygen atoms, and combinations thereof. A spacer moiety may also beinterposed, e.g., between a water-soluble polymer and a terminal portionof the overall polymer reagent structure, wherein the terminal portionis typically where covalent attachment to EPO occurs. The spacer moietycan comprise an amide, secondary amine, carbamate, thioether, ordisulfide group. Nonlimiting examples of specific spacer moietiesinclude those selected from the group consisting of —O—, —S—, —S—S—,—C(O)—, —C(O)—NH—, —NH—C(O)—NH—, —O—C(O)—NH—, —C(S)—, —CH₂—, —CH₂—CH₂—,—CH₂—CH₂—CH₂—, —CH₂—CH₂—CH₂—CH₂—, —O—CH₂—, —CH₂—O—, —O—CH₂—CH₂—,—CH₂—O—CH₂—, —CH₂—CH₂—O—, —O—CH₂—CH₂—CH₂—, —CH₂—O—CH₂—CH₂—,—CH₂—CH₂—O—CH₂—, —CH₂—CH₂—CH₂—O—, —O—CH₂—CH₂—CH₂—CH₂—,—CH₂—O—CH₂—CH₂—CH₂—, —CH₂—CH₂—O—CH₂—CH₂—, —CH₂—CH₂—CH₂—O—CH₂—,—CH₂—CH₂—CH₂—CH₂—O—, —C(O)—NH—CH₂—, —C(O)—NH—CH₂—CH₂—,—CH₂—C(O)—NH—CH₂—, —CH₂—CH₂—C(O)—NH—, —C(O)—NH—CH₂—CH₂—CH₂—,—CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—C(O)—NH—CH₂—, —CH₂—CH₂—CH₂—C(O)—NH—,—C(O)—NH—CH₂—CH₂—CH₂—CH₂—, —CH₂—C(O)—NH—CH₂—CH₂—CH₂—,—CH₂—CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—CH₂—CH₂—C(O)—NH—, —C(O)—O—CH₂—,—CH₂—C(O)—O—CH₂—, —CH₂—CH₂—C(O)—O—CH₂—, —C(O)—O—CH₂—CH₂—, —NH—C(O)—CH₂—,—CH₂—NH—C(O)—CH₂—, —CH₂—CH₂—NH—C(O)—CH₂—, —NH—C(O)—CH₂—CH₂—,—CH₂—NH—C(O)—CH₂—CH₂—, —CH₂—CH₂—NH—C(O)—CH₂—CH₂—, —C(O)—NH—CH₂—,—C(O)—NH—CH₂—CH₂—, —O—C(O)—NH—CH₂—, —O—C(O)—NH—CH₂—CH₂—, —NH—CH₂—,—NH—CH₂—CH₂—, —CH₂—NH—CH₂—, —CH₂—CH₂—NH—CH₂—, —C(O)—CH₂—,—C(O)—CH₂—CH₂—, —CH₂—C(O)—CH₂—, —CH₂—CH₂—C(O)—CH₂—,—CH₂—CH₂—C(O)—CH₂—CH₂—, —CH₂—CH₂—C(O)—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—CH₂—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—CH₂—CH₂—,—O—C(O)—NH—[CH₂]_(h)—(OCH₂CH₂)_(j)—, bivalent cycloalkyl group, —O—,—S—, an amino acid, —N(R⁶)—, and combinations of two or more of any ofthe foregoing, wherein R⁶ is H or an organic radical selected from thegroup consisting of alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, aryl and substituted aryl, (h) iszero to six, and (j) is zero to 20. Other specific spacer moieties havethe following structures: —C(O)—NH—(CH₂)₁₋₆—NH—C(O)—,—NH—C(O)—NH—(CH₂)₁₋₆—NH—C(O)—, and —O—C(O)—NH—(CH₂)₁₋₆—NH—C(O)—, whereinthe subscript values following each methylene indicate the number ofmethylenes contained in the structure, e.g., (CH₂)₁₋₆ means that thestructure can contain 1, 2, 3, 4, 5 or 6 methylenes. Additionally, anyof the above spacer moieties may further include an ethylene oxideoligomer chain comprising 1 to 20 ethylene oxide monomer units [i.e.,—(CH₂CH₂O)₁₋₂₀]. That is, the ethylene oxide oligomer chain can occurbefore or after the spacer moiety, and optionally in between any twoatoms of a spacer moiety comprised of two or more atoms. Also, theoligomer chain would not be considered part of the spacer moiety if theoligomer is adjacent to a polymer segment and merely represent anextension of the polymer segment.

Additional exemplary EPO polymer conjugates and polymer reagents inaccordance with one or more embodiments of the invention are providedbelow, and are additionally described in the Examples which follow.

One exemplary conjugate of erythropoietin (EPO) possesses the structure:

wherein POLY is a polyalkylene oxide, Q is an optional linking grouphaving a length of from one to 10 atoms, m is an integer ranging from 0to 20, Z is selected from the group consisting of alkyl, substitutedalkyl, aryl and substituted aryl, EPO is a residue of erythropoietin,and “—NH-EPO” represents an amino group of EPO.

In reference to the structure above, Z refers to a substituent on thecarbon alpha to the carbonyl group. The placement of the substituent inthe α-position provides additional selectivity to the reagent, and thus,in the resulting conjugate. Preferably, Z is lower alkyl or substitutedlower alkyl, e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, orisobutyl. Most preferably, Z is methyl.

An illustrative EPO conjugate structure falling within the generalizedstructure provided above comprises:

where n typically ranges from about 200 to about 1400.

Also provided in one or more embodiments of the invention are EPOconjugates corresponding to one or more of the following structures:

where n corresponds to the values previously described and EPO is aresidue of an EPO moiety.

Compositions

The conjugates typically form part of a composition. Generally, thecomposition comprises a plurality of conjugates, preferably although notnecessarily, each conjugate is comprised of the same EPO moiety (i.e.,within the entire composition, only one type of EPO moiety is found). Inaddition, the composition can comprise a plurality of conjugates whereinany given conjugate is comprised of a moiety selected from the groupconsisting of two or more different EPO moieties (i.e., within theentire composition, two or more different EPO moieties are found).Optimally, however, substantially all of the plurality of conjugates inthe composition (e.g., 85% or more of the plurality of conjugates in thecomposition) are each comprised of the same EPO moiety.

The composition can comprise a single conjugate species (e.g., amonoPEGylated conjugate wherein a single polymer is attached at the samelocation or position on EPO for substantially all conjugates in thecomposition) or a mixture of conjugate species (e.g., a mixture ofmonoPEGylated conjugates where attachment of the polymer occurs atdifferent sites and/or a mixture of monoPEGylated, diPEGylated andtriPEGylated, etc., conjugates). The compositions can also compriseother conjugates having four, five, six, seven, eight or more polymersattached to any given moiety having EPO activity. In addition, theinvention includes instances wherein the composition comprises aplurality of conjugates, each conjugate comprising one non-peptidicwater-soluble polymer covalently attached to one EPO moiety, as well ascompositions comprising two, three, four, five, six, seven, eight, ormore water-soluble polymers covalently attached to one EPO moiety.

In one or more embodiments, it is preferred that theconjugate-containing composition is free or substantially free ofalbumin. It is also preferred that the composition is free orsubstantially free of proteins that do not have EPO activity. Thus, itis preferred that the composition is 85%, more preferably 95%, and mostpreferably 99% free of albumin. Additionally, it is preferred that thecomposition is 85%, more preferably 95%, and most preferably 99% free ofany protein that does not have EPO activity.

Control of the desired number of polymers for any given moiety can beachieved by selecting the proper polymeric reagent, the ratio ofpolymeric reagent to the EPO moiety, temperature, pH conditions, andother aspects of the conjugation reaction. In addition, reduction orelimination of the undesired conjugates (e.g., those conjugates havingfour or more attached polymers) can be achieved through purification.

For example, the polymer-EPO moiety conjugates can be purified toobtain/isolate different conjugated species. Specifically, the productmixture can be purified to obtain an average of anywhere from one, two,three, four, five or more PEGs per EPO moiety, typically one, two orthree PEGs per EPO moiety. The strategy for purification of the finalconjugate reaction mixture will depend upon a number of factors,including, for example, the molecular weight of the polymeric reagentemployed, the particular EPO moiety, the desired dosing regimen, and theresidual activity and in vivo properties of the individual conjugate(s).

Exemplary compositions in accordance with the invention are thosecomprising monoPEGylated EPO, diPEGylated EPO, or mixtures thereof.Illustrative compositions include the following: an EPO conjugatecomposition wherein greater than about 85% of the PEG-EPO conjugates aremonoPEGylated EPO, or an EPO conjugate composition wherein greater thanabout 90%, or even about 95% or greater of the PEG-EPO conjugates aremonoPEGylated EPO.

If desired, conjugates having different molecular weights can beisolated using gel filtration chromatography and/or ion exchangechromatography. That is to say, gel filtration chromatography is used tofractionate differently numbered polymer-to-EPO moiety ratios (e.g.,1-mer, 2-mer, 3-mer, and so forth, wherein “1-mer” indicates 1 polymerto EPO moiety, “2-mer” indicates two polymers to EPO moiety, and so on)on the basis of their differing molecular weights (where the differencecorresponds essentially to the average molecular weight of thewater-soluble polymer portion). For example, in an exemplary reactionwhere a 35,000 Dalton protein is randomly conjugated to a polymericreagent having a molecular weight of about 20,000 Daltons, the resultingreaction mixture may contain unmodified protein (having a molecularweight of about 35,000 Daltons), monoPEGylated protein (having amolecular weight of about 55,000 Daltons), diPEGylated protein (having amolecular weight of about 75,000 Daltons), and so forth.

While this approach can be used to separate PEG and other polymer-EPOmoiety conjugates having different molecular weights, this approach isgenerally ineffective for separating positional isoforms havingdifferent polymer attachment sites within the EPO moiety. For example,gel filtration chromatography can be used to separate from each othermixtures of PEG 1-mers, 2-mers, 3-mers, and so forth, although each ofthe recovered conjugate compositions may contain PEG(s) attached todifferent reactive groups (e.g., lysine residues) within the EPO moiety.

Gel filtration columns suitable for carrying out this type of separationinclude Superdex™ and Sephadex™ columns available from AmershamBiosciences (Piscataway, N.J.). Selection of a particular column willdepend upon the desired fractionation range desired. Elution isgenerally carried out using a suitable buffer, such as phosphate,acetate, or the like. The collected fractions may be analyzed by anumber of different methods, for example, (i) absorbance at 280 nm forprotein content, (ii) dye-based protein analysis using bovine serumalbumin (BSA) as a standard, (iii) iodine testing for PEG content (Simset al. (1980) Anal. Biochem, 107:60-63), (iv) sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS PAGE), followed by staining withbarium iodide, and (v) high performance liquid chromatography (HPLC).

Separation of positional isoforms is carried out by reverse phase-highperformance liquid chromatography (RP-HPLC) using a suitable column(e.g., a C18 column or C3 column, available commercially from companiessuch as Amersham Biosciences or Vydac) or by ion exchange chromatographyusing an ion exchange column, e.g., a Sepharose™ ion exchange columnavailable from Amersham Biosciences. Either approach can be used toseparate polymer-EPO isomers having the same molecular weight (i.e.,positional isoforms).

The compositions are preferably substantially free of proteins that donot have EPO activity. In addition, the compositions preferably aresubstantially free of all other noncovalently attached water-solublepolymers. In some circumstances, however, the composition can contain amixture of polymer-EPO moiety conjugates and unconjugated EPO moiety.

Optionally, the composition of the invention further comprises apharmaceutically acceptable excipient. If desired, the pharmaceuticallyacceptable excipient can be added to a conjugate to form such acomposition.

Exemplary excipients include, without limitation, those selected fromthe group consisting of carbohydrates, inorganic salts, antimicrobialagents, antioxidants, surfactants, buffers, acids, bases, andcombinations thereof.

A carbohydrate such as a sugar, a derivatized sugar such as an alditol,aldonic acid, an esterified sugar, and/or a sugar polymer may be presentas an excipient. Specific carbohydrate excipients include, for example:monosaccharides, such as fructose, maltose, galactose, glucose,D-mannose, sorbose, and the like; disaccharides, such as lactose,sucrose, trehalose, cellobiose, and the like; polysaccharides, such asraffinose, melezitose, maltodextrins, dextrans, starches, and the like;and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol,sorbitol (glucitol), pyranosyl sorbitol, myoinositol, and the like.

The excipient can also include an inorganic salt or buffer such ascitric acid, sodium chloride, potassium chloride, sodium sulfate,potassium nitrate, sodium phosphate monobasic, sodium phosphate dibasic,and combinations thereof.

The composition can also include an antimicrobial agent for preventingor deterring microbial growth. Nonlimiting examples of antimicrobialagents suitable for one or more embodiments of the present inventioninclude benzalkonium chloride, benzethonium chloride, benzyl alcohol,cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol,phenylmercuric nitrate, thimersol, and combinations thereof.

An antioxidant can be present in the composition as well. Antioxidantsare used to prevent oxidation, thereby preventing the deterioration ofthe conjugate or other components of the preparation. Suitableantioxidants for use in one or more embodiments of the present inventioninclude, for example, ascorbyl palmitate, butylated hydroxyanisole,butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propylgallate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodiummetabisulfite, and combinations thereof.

A surfactant can be present as an excipient. Exemplary surfactantsinclude: polysorbates, such as “Tween 20” and “Tween 80,” and pluronicssuch as F68 and F88 (both of which are available from BASF, Mount Olive,N.J.); sorbitan esters; lipids, such as phospholipids such as lecithinand other phosphatidylcholines, phosphatidylethanolamines (althoughpreferably not in liposomal form), fatty acids and fatty esters;steroids, such as cholesterol; and chelating agents, such as EDTA, zincand other such suitable cations.

Acids or bases can be present as an excipient in the composition.Nonlimiting examples of acids that can be used include those acidsselected from the group consisting of hydrochloric acid, acetic acid,phosphoric acid, citric acid, malic acid, lactic acid, formic acid,trichloroacetic acid, nitric acid, perchloric acid, phosphoric acid,sulfuric acid, fumaric acid, and combinations thereof. Examples ofsuitable bases include, without limitation, bases selected from thegroup consisting of sodium hydroxide, sodium acetate, ammoniumhydroxide, potassium hydroxide, ammonium acetate, potassium acetate,sodium phosphate, potassium phosphate, sodium citrate, sodium formate,sodium sulfate, potassium sulfate, potassium fumerate, and combinationsthereof.

The amount of the conjugate (i.e., the conjugate formed between theactive agent and the polymeric reagent) in the composition will varydepending on a number of factors, but will optimally be atherapeutically effective dose when the composition is stored in a unitdose container (e.g., a vial). In addition, the pharmaceuticalpreparation can be housed in a syringe. A therapeutically effective dosecan be determined experimentally by repeated administration ofincreasing amounts of the conjugate in order to determine which amountproduces a clinically desired endpoint. Typically, a pharmaceuticalcomposition of the invention will contain different amounts of an EPOmoiety, e.g., from about 10 to about 10,000 μg/ml EPO conjugate,preferably from about 50 μg/ml to about 400 μg/ml EPO conjugate.

The amount of any individual excipient in the composition will varydepending on the activity of the excipient and particular needs of thecomposition. Typically, the optimal amount of any individual excipientis determined through routine experimentation, i.e., by preparingcompositions containing varying amounts of the excipient (ranging fromlow to high), examining the stability and other parameters, and thendetermining the range at which optimal performance is attained with nosignificant adverse effects.

Generally, however, the excipient will be present in the composition inan amount of about 1% to about 99% by weight, preferably from about 5%to about 98% by weight, more preferably from about 15 to about 95% byweight of the excipient, with concentrations less than 30% by weightmost preferred.

These foregoing pharmaceutical excipients along with other excipientsare described in “Remington: The Science & Practice of Pharmacy”,19^(th) ed., Williams & Williams, (1995), the “Physician's DeskReference”, ed., Medical Economics, Montvale, N.J. (1998), and Kibbe, A.H., Handbook of Pharmaceutical Excipients, 3^(rd) Edition, AmericanPharmaceutical Association, Washington, D.C., 2000.

The compositions encompass all types of formulations and in particularthose that are suited for injection, e.g., powders or lyophilates thatcan be reconstituted as well as liquids. Examples of suitable diluentsfor reconstituting solid compositions prior to injection includebacteriostatic water for injection, dextrose 5% in water,phosphate-buffered saline, Ringer's solution, saline, sterile water,deionized water, and combinations thereof. With respect to liquidpharmaceutical compositions, solutions and suspensions are envisioned.

The compositions of one or more embodiments of the present invention aretypically, although not necessarily, administered via injection and aretherefore generally liquid solutions or suspensions immediately prior toadministration. The pharmaceutical preparation can also take other formssuch as syrups, creams, ointments, tablets, powders, and the like. Othermodes of administration are also included, such as pulmonary, rectal,transdermal, transmucosal, oral, intrathecal, subcutaneous,intra-arterial, and so forth.

Administration

The invention also provides a method for administering a conjugate asprovided herein to a patient suffering from a condition that isresponsive to treatment with the conjugate. The method comprisesadministering, generally via injection, a therapeutically effectiveamount of the conjugate (preferably provided as part of a pharmaceuticalcomposition). As previously described, the conjugates can beadministered parenterally by intravenous injection, or less preferablyby intramuscular or by subcutaneous injection. Suitable formulationtypes for parenteral administration include ready-for-injectionsolutions, dry powders for combination with a solvent prior to use,suspensions ready for injection, dry insoluble compositions forcombination with a vehicle prior to use, and emulsions and liquidconcentrates for dilution prior to administration, among others.

The method of administering may be used to treat any condition that canbe remedied or prevented by administration of the conjugate. Those ofordinary skill in the art appreciate which conditions a specificconjugate can effectively treat. For example, an EPO conjugate of theinvention can be used to treat patients suffering from anemia. Exemplaryanemies include (a) anemia associated with chronic renal failure, (b)anemia related to zidovudine therapy in HIV-infected patients, and (c)anemia in cancer patients undergoing chemotherapy. In addition, theconjugates can be used to reduce allogeneic blood transfusion in surgerypatients. Advantageously, the conjugate can be administered to thepatient prior to, simultaneously with, or after administration ofanother active agent, such as a chemotherapy agent. In addition, theconjugate can be administered to an anemic patient prior to undergoingsurgery.

The actual dose to be administered will vary depending upon the age,weight, and general condition of the subject as well as the severity ofthe condition being treated, the judgment of the health careprofessional, and conjugate being administered. Therapeuticallyeffective amounts are known to those skilled in the art and/or aredescribed in the pertinent reference texts and literature. Generally, atherapeutically effective amount will range from about 0.001 mg to 100mg, preferably in doses from 0.01 mg/day to 75 mg/day, and morepreferably in doses from 0.10 mg/day to 50 mg/day. For example, aconjugate of the invention may be administered at 0.01 to 10 μg perkilogram body weight, preferably 0.1 to 3 μg per kilogram body weight,e.g., once daily, every other day, twice weekly, once weekly, once everyother week, or as described below.

The unit dosage of any given conjugate (again, preferably provided aspart of a pharmaceutical preparation) can be administered in a varietyof dosing schedules depending on the judgment of the clinician, needs ofthe patient, and so forth. The specific dosing schedule will be known bythose of ordinary skill in the art or can be determined experimentallyusing routine methods. Exemplary dosing schedules include, withoutlimitation, administration five times a day, four times a day, threetimes a day, twice daily, once daily, three times weekly, twice weekly,once weekly, twice monthly, once monthly, and any combination thereof.Once the clinical endpoint has been achieved, dosing of the compositionis halted. Particularly preferred compositions are those that are dosedat a frequency of about once a week or less. As seen in the accompanyingExamples, a preferred EPO conjugate, mono-mPEG-SMB-30kD-EPO, was shownin vivo to sustain higher blood levels over an extended period of timethan either the unmodified EPO control, or a currently marketed versionof EPO.

One advantage of administering certain conjugates described herein isthat individual water-soluble polymer portions can be cleaved. Such aresult is advantageous when clearance from the body is potentially aproblem because of the polymer size. Optimally, cleavage of eachwater-soluble polymer portion is facilitated through the use ofphysiologically cleavable and/or enzymatically degradable linkages suchas amide, carbonate or ester-containing linkages. In this way, clearanceof the conjugate (via cleavage of individual water-soluble polymerportions) can be modulated by selecting the polymer molecular size andthe type functional group that would provide the desired clearanceproperties. One of ordinary skill in the art can determine the propermolecular size of the polymer as well as the cleavable functional group.For example, one of ordinary skill in the art, using routineexperimentation, can determine a proper molecular size and cleavablefunctional group by first preparing a variety of polymer derivativeswith different polymer weights and cleavable functional groups, and thenobtaining the clearance profile (e.g., through periodic blood or urinesampling) by administering the polymer derivative to a patient andtaking periodic blood and/or urine sampling. Once a series of clearanceprofiles have been obtained for each tested conjugate, a suitableconjugate can be identified.

It is to be understood that while the invention has been described inconjunction with the preferred specific embodiments thereof, that theforegoing description as well as the examples that follow are intendedto illustrate and not limit the scope of the invention. Other aspects,advantages and modifications within the scope of the invention will beapparent to those skilled in the art to which the invention pertains.

All articles, books, patents and other publications referenced hereinare hereby incorporated by reference in their entireties.

EXPERIMENTAL

The practice of the invention will employ, unless otherwise indicated,conventional techniques of organic synthesis, biochemistry, proteinpurification and the like, which are within the skill of the art. Suchtechniques are fully explained in the literature. See, for example, J.March, Advanced Organic Chemistry: Reactions Mechanisms and Structure,4th Ed. (New York: Wiley-Interscience, 1992), supra. Reagents andmaterials are commercially available unless specifically stated to thecontrary.

In the following examples, efforts have been made to ensure accuracywith respect to numbers used (e.g., amounts, temperatures, etc.) butsome experimental error and deviation should be accounted for. Unlessindicated otherwise, temperature is in degrees C. and pressure is at ornear atmospheric pressure at sea level. Each of the following examplesis considered to be instructive to one of ordinary skill in the art forcarrying out one or more of the embodiments described herein.

Although other abbreviations known by one having ordinary skill in theart will be referenced, other reagents and materials will be used, andother methods known by one having ordinary skill in the art will beused, the following list and methods description is provided for thesake of convenience.

ABBREVIATIONS

mPEG-SPA mPEG-succinimidyl propionate

mPEG-SBA mPEG-succinimidyl butanoate

mPEG-MAL mPEG-maleimide, CH₃O—(CH₂CH₂O)_(n)—CH₂CH₂-MAL

mPEG-SMP mPEG-succinimidyl α-methylpropanoate,CH₃O—(CH₂CH₂O)_(n)—CH₂—CH(CH₃)—C(O)—O-succinimide

mPEG-SMB mPEG-succinimidyl α-methylbutanoate,CH₃O—(CH₂CH₂O)_(n)—CH₂CH₂—CH(CH₃)—C(O)—O-succinimide

anh. Anhydrous

Fmoc 9-fluorenylmethoxycarbonyl

NaCNBH₃ sodium cyanoborohydride

HCl hydrochloric acid

NMR nuclear magnetic resonance

DI deionized

MW molecular weight

K or kDa kilodaltons

IEX ion exchange

SEC Size exclusion chromatography

HPLC high performance liquid chromatography

FPLC fast protein liquid chromatography

SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis

MALDI-TOF Matrix Assisted Laser Desorption Ionization, Time-of-Flight

TLC Thin Layer Chromatography

EPO corresponding to the amino acid sequence of SEQ. ID. NO. 1. was usedin Examples 1-10. The EPO stock solution contained about 0.5 mg/mL to2.1 mg/mL of EPO (depending on the lot) in an amine-free buffer.Polymeric reagents are available from Nektar Therapeutics, Huntsville,Ala., unless indicated otherwise.

Sample Analysis

Samples were analyzed by RP-HPLC (Reverse Phase) and purified using IEX(Ion Exchange) chromatography. RP-HPLC was also used to prepare standardcurves to evaluate protein and conjugate concentrations in variouspreparations. The Bradford Protein Assay (Bradford, M M. AnalyticalBiochemistry 72: 248-254, 1976) can also be used to determine proteincontent.

SDS-PAGE Analysis

Certain samples as indicated were analyzed using SDS-PAGE for sampledetection only. Samples were analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using Sure-Lock II(Invitrogen). Samples were mixed with sample buffer. Then, the preparedsamples were loaded onto a gel and run for approximately 30 minutes.

Example 1 Random PEGylation of EPO with mPEG-SMB, 30 kDa

mPEG-SMB, 30 kDa, stored at −20° C. under argon, was warmed to ambienttemperature. The warmed mPEG-SMB (20.0 mg) was dissolved in 1.0 mL of 2mM HCl to form an mPEG-SMB solution. The mPEG-SMB solution was added toa previously prepared EPO reaction mixture (500 μL stock EPO solution,500 μL of 20 mM NaPO₄, pH 6.5) until a twenty-fold molar excess ofmPEG-SMB relative to EPO was reached. After the addition of themPEG-SMB, the pH of the reaction mixture was readjusted to 6.5 and wasmixed well. To allow for coupling of the mPEG-SMB to EPO via an amidelinkage, the reaction solution was stirred for six hours at roomtemperature and thereafter stirred for twelve hours at 3-8° C. in a coldroom, thereby resulting in a conjugate solution. The reaction wasquenched by addition of glycine.

The conjugate solution was purified using ion exchange chromatography(anion-strong). SDS-PAGE and RP-HPLC (C₃) analysis was also used for thecharacterization. Based upon the HPLC and SDS-PAGE results, the overallPEGylation yield was ˜45%, meaning that approximately 45% of the EPO wasconjugated to PEG. The reaction mixture contained a mixture of PEG-mers(monoPEGylated EPO, diPEGylated EPO, etc.), as well as unreacted EPO.The mixture of PEGmers was then resolved to provide a purified monoPEGconjugate that was substantially (>90%) monoPEGylated EPO (i.e., EPO1-mer).

Using this same approach, other conjugates can be prepared usingmPEG-SMB having other weight average molecular weights.

Example 2 Random PEGylation of EPO with branchedmPEG2-N-Hydroxysuccinimide, 40 kDa

Branched mPEG2-N-Hydroxysuccinimide (NHS), 40 kDa

Branched mPEG2-N-hydroxysuccinimide, 40 kDa, stored at −20° C. underargon, was warmed to ambient temperature. The branchedmPEG2-N-hydroxysuccinimide (26.7 mg) was dissolved in 1.0 mL of 2 mM HClto form a branched mPEG2-N-hydroxy-succinimide solution. The branchedmPEG2-N-hydroxysuccinimide solution was added to a previously preparedEPO reaction mixture (500 μL stock EPO solution, 5004 of 20 mM NaPO₄)until a twenty-fold molar excess of branched mPEG2-N-hydroxysuccinimidesolution relative to EPO was reached. After the addition of branchedmPEG2-N-hydroxysuccinimide, the pH of the reaction mixture wasreadjusted to 6.5 and was mixed well. To allow for coupling of thebranched mPEG2-N-hydroxysuccinimide to EPO via an amide linkage, thereaction solution was stirred for six hours at room temperature andthereafter stirred for twelve hours at 3-8° C. in a cold room, therebyresulting in a conjugate solution. The reaction was quenched withglycine.

The conjugate solution was purified using ion exchange chromatography(anion-strong). SDS-PAGE and RP-HPLC (C₃) analysis were also used forthe characterization. Based upon the HPLC and SDS-PAGE results, theoverall PEGylation yield was ˜32%.

The reaction mixture contained a mixture of PEG-mers (monoPEGylated EPO,diPEGylated EPO, etc.), as well as unreacted EPO. The mixture of PEGmerswas then resolved to provide a purified monoPEG conjugate that wassubstantially (>90%) monoPEGylated EPO (i.e., EPO 1-mer).

Using this same approach, other conjugates can be prepared usingbranched mPEG2-N-hydroxysuccinimide having other weight averagemolecular weights.

Example 3 Random PEGylation of EPO with mPEG-SMB, 30 kDa

mPEG-SMB, 30 kDa, stored at −20° C. under argon, was warmed to ambienttemperature. Two 10.0 mg samples of the mPEG-SMB were separatelydissolved in 500 μL of 2 mM HCl to form two aliquots of mPEG-SMBsolution. A first aliquot of mPEG-SMB solution was added to a previouslyprepared EPO reaction mixture (250 μL stock EPO solution, 20 mM NaPO₄,pH 6.5) until a twenty-fold molar excess of mPEG-SMB relative to EPO wasreached. The pH was tested and adjusted as necessary to ensure a pH of6.5. After about thirty minutes, the second aliquot of mPEG-SMB solutionwas added and mixed well, thereby resulting in a reaction mixture havinga forty-fold molar excess of mPEG-SMB relative to EPO. Again, the pH wastested and adjusted as necessary to ensure a pH of about 6.5. To allowfor final coupling of the mPEG-SMB to EPO via an amide linkage, thereaction solution was stirred for sixteen hours at 3-8° C. in a coldroom, thereby resulting in a conjugate solution. The reaction wasquenched by addition of glycine.

The conjugate solution was purified using ion exchange chromatography(anion-strong). SDS-PAGE and RP-HPLC (C₃) analysis were also used forthe characterization. Based upon the HPLC and SDS-PAGE results, theoverall PEGylation yield was ˜62.5% (representing about ˜54%monoPEGylated and ˜8.5% diPEGylated conjugates). The reaction yield wasimproved over that reported in Example 1 by increasing both the overallamount of PEG reagent used and the manner of its addition.

The above reaction was run again under nearly identical reactionconditions, with the exception that the pH was adjusted to approximately7.5 after addition of each aliquot of mPEG-SMB. The resulting conjugatesolution was purified using ion exchange chromatography as describedabove. The purified monoPEGylated product, designated 01-R-MSMBE-30, wasdetermined by SDS-PAGE to contain approximately 94% monomer(mono-mPEG-SMB-30kD-EPO) and 6% dimer (di-mPEG-SMB-30kD-EPO). NotriPEGylated product was detected. The purified diPEGylated product,designated 01-R-DSMBE-30, was determined by SDS-PAGE to containapproximately 67% dimer (di-mPEG-SMB-30kD-EPO) and 28% monomer(monomPEG-SMB-30kD-EPO). No triPEGylated product was detected. Furtherseparations can be carried out as desired to arrive at a compositionthat is substantially pure dimer.

MALDI-TOF analysis for both the mono- and diPEGylated species confirmedthe covalent attachment of either one PEG moiety or two PEG moieties,respectively, to form the respective conjugate products.

Preliminary in vivo and in vitro assays were conducted on the purifiedconjugates, mono-mPEG-SMB-30kD-EPO and di-mPEG-SMB-30kD-EPO. The resultsare provided below. The assays confirmed the bioactivity of the subjectconjugates.

TABLE 4 Conjugate In vivo Activity, IU/mg* In vitro Assay**mono-mPEG-SMB- 507500 1.9% 30kD-EPO) Sample 01-R-MSMBE-30di-mPEG-SMB-30kD-EPO 108500 0.9% Sample 01-R-DSMBE-30 *unmodified EPOpossesses an activity of 120,000 IU/mg in the assay set up employed.**for comparison, unmodified EPO is considered to possess an activity of100%.

Using this same approach, other conjugates can be prepared usingmPEG-SMB having other weight average molecular weights.

Example 4 Random PEGylation of EPO with BranchedmPEG2-N-Hydroxysuccinimide, 40 kDa

Branched mPEG2-N-Hydroxysuccinimide, 40 kDa

Branched mPEG2-N-hydroxysuccinimide, 40 kDa, stored at −20° C. underargon, was warmed to ambient temperature. Two 13.3 mg samples of thebranched mPEG2-N-hydroxysuccinimide were separately dissolved in 250 μLof 2 mM HCl to form two aliquots of mPEG2-N-hydroxysuccinimide solution.A first aliquot of branched mPEG2-N-hydroxysuccinimide solution wasadded to a previously prepared EPO reaction mixture (250 μL stock EPOsolution, 20 mM NaPO₄, pH 6.5) until a twenty-fold molar excess ofmPEG2-N-hydroxysuccinimide to EPO was reached. The pH was tested andadjusted as necessary to ensure a pH of 6.5. After about thirty minutes,the second aliquot of branched mPEG2-N-hydroxysuccinimide solution wasadded and mixed well, thereby resulting in a reaction mixture havingforty-fold molar excess of branched mPEG2-N-hydroxysuccinimide relativeto EPO. Again, the pH was tested and adjusted as necessary to ensure apH of 6.5. To allow for final coupling of the branchedmPEG2-N-hydroxysuccinimide to EPO via an amide linkage, the reactionsolution was stirred for sixteen hours at 3-8° C. in a cold room,thereby resulting in a conjugate solution. The reaction was quenched byaddition of glycine.

SDS-PAGE analysis was used for the characterization. Based upon theSDS-PAGE results, the PEGylation yield was ˜70% (representing ˜64%monoPEGylated and ˜6% diPEGylated conjugates). By increasing both theamount of PEG used and the manner of its addition, the PEGylation yieldwas increased over that described in Example 2.

Using this same approach, other conjugates can be prepared usingbranched mPEG2-N-hydroxysuccinimide having other weight averagemolecular weights.

Example 5 Random PEGylation of EPO with mPEG-SMB, 30 kDa

In this reaction, the hydrophobicity of the buffer system was altered byaddition of ethanol. Ethanol was added to 250 μL of a previouslyprepared EPO reaction mixture (250 μL stock EPO solution, 500 μL 20 mMNaPO₄) to form a 10% ethanol-containing EPO reaction mixture.Separately, mPEG-SMB, 30 kDa, stored at −20° C. under argon, was warmedto ambient temperature. The mPEG-SMB (13.3 mg) was dissolved in 250 μLof 2 mM HCl to form an mPEG-SMB solution. To the ethanol-containing EPOreaction mixture, the mPEG-SMB solution was added until a twenty-foldmolar excess of mPEG-SMB relative to EPO was reached. The pH was testedand adjusted as necessary to ensure a pH of 6.5. After about thirtyminutes, 13.3 mg of dry mPEG-SMB was added and mixed well, therebyresulting in a reaction mixture having forty-fold molar excess ofmPEG-SMB relative to EPO. Again, the pH was tested and adjusted asnecessary to ensure a pH of 6.5. To allow for final coupling of themPEG-SMB to EPO via an amide linkage, the reaction solution was stirredfor sixteen hours at 3-8° C. in a cold room, thereby resulting in aconjugate solution. The reaction was quenched by addition of glycine.

SDS-PAGE analysis was used for characterization of the reaction mixture.Based upon the SDS-PAGE results, the PEGylation yield was ˜35%(representing ˜28% monoPEGylated and ˜7.0% diPEGylated conjugates).

Using this same approach, other conjugates can be prepared usingmPEG-SMB having other weight average molecular weights.

Example 6 Random PEGylation of EPO with BranchedmPEG2-N-Hydroxysuccinimide, 40 kDa

Branched mPEG2-N-Hydroxysuccinimide, 40 kDa

In this reaction, the hydrophobicity of the buffer system was altered byaddition of ethanol. Ethanol was added to 250 μL of a previouslyprepared EPO reaction mixture (250 μL stock EPO solution, 500 μL 20 mMNaPO₄) to form a 10% ethanol-containing EPO reaction mixture. BranchedmPEG2-N-hydroxysuccinimide, 40 kDa, stored at −20° C. under argon, waswarmed to ambient temperature. Two 13.3 mg samples of the warmedbranched mPEG2-N-hydroxysuccinimide were separately dissolved in 250 μl,of 2 mM HCl to form two aliquots of mPEG2-N-hydroxysuccinimide solution.A first aliquot of branched mPEG2-N-hydroxysuccinimide solution wasadded to the 10% ethanol-containing EPO reaction mixture until atwenty-fold molar excess of mPEG2-N-hydroxysuccinimide relative to EPOwas reached. The pH was tested and adjusted as necessary to ensure a pHof 6.5. After about thirty minutes, the second aliquot of branchedmPEG2-N-hydroxysuccinimide solution was added and mixed well, therebyresulting in a reaction mixture having forty-fold molar excess ofbranched mPEG2-N-hydroxysuccinimide relative to EPO. Again, the pH wastested and adjusted as necessary to ensure a pH of 6.5. To allow forfinal coupling of the branched mPEG2-N-hydroxysuccinimide to EPO via anamide linkage, the reaction solution was stirred for 16 hours at 3-8° C.in a cold room, thereby resulting in a conjugate solution. The reactionwas quenched by addition of glycine.

SDS-PAGE analysis was used for characterization of the reaction mixture.Based upon the SDS-PAGE results, the PEGylation yield was ˜27%(representing ˜21% monoPEGylated and ˜6.0% diPEGylated conjugates).

Using this same approach, other conjugates can be prepared usingbranched mPEG2-N-hydroxysuccinimide having other weight averagemolecular weights.

Example 7 Non-Random PEGylation of EPO with BranchedmPEG2-N-Hydroxysuccinimide, 40 kDa

Branched mPEG2-N-Hydroxysuccinimide, 40 kDa

Prior to the conjugation reaction, 1.5 mL of reaction buffer (20 mMNaPO₄), pH 8.0, was added to 500 μL of stock EPO solution. To reversiblyprotect the most reactive amino groups in EPO (e.g., those aminesassociated with the Lys-52 side chain), the EPO solution was adjusted topH 8.0 and then combined with a 10-fold molar excess of dimethylmaleicanhydride, “DMMAn”, (Tsunoda, S., et al., J. Pharmacol. Exp. Ther. 1999,290, 368-72) relative to the lysine amino acids in EPO, to thereby forma DMMAn-treated EPO solution. The pH was tested and adjusted asnecessary to ensure a pH of 8.0.

Branched mPEG2-N-hydroxysuccinimide, 40 kDa, stored at −20° C. underargon, was warmed to ambient temperature. The branchedmPEG2-N-hydroxysuccinimide, 40 kDa, (26.7 mg) was dissolved in 1.0 mL of2 mM HCl to form a branched mPEG2-N-hydroxysuccinimide solution. Thebranched mPEG2-N-hydroxysuccinimide solution was added to theDMMAn-treated EPO solution (pH 8.0, room temperature), until atwenty-fold molar excess of branched mPEG2-N-hydroxysuccinimide relativeto EPO was reached. To allow for coupling of the branchedmPEG2-N-hydroxysuccinimide to EPO via an amide linkage, the reactionsolution was stirred for two hours at room temperature and thereafterstirred for fourteen hours at 3-8° C. in a cold room, thereby resultingin a conjugate solution. The reaction was quenched by addition ofglycine. Thereafter, to deprotect the protected lysine amino groups, thereaction mixture was adjusted to pH 6.0 with 0.1 N HCl and incubated at37° C. for 30 minutes.

SDS-PAGE analysis was used for characterization. Based upon the SDS-PAGEresults, the PEGylation yield of monoPEGylated conjugate (EPO 1-mer) was˜20%. It is believed that the DMMan preferentially reacted with theamino groups of Lys-52, thereby promoting conjugation of the mPEG-2-NHSreagent at sites other than Lys-52. The conjugate composition waspurified as previously described.

Based on RP-HPLC analysis, the purified monomer composition, designatedherein as 01-P-MBNHS-40, contained 95+% monoPEGylatedEPO conjugate, andless than 5% diPEGylated EPO conjugate. The purified dimer composition,designated herein as 01-P-DBNHS-40, contained essentially purediPEGylated EPO conjugate (RP-HPLC).

Based upon the reaction protocol used, it is believed that a majority ofthe conjugate species present in the resulting product mixture possessPEG covalently attached to a site other than the Lys-52, and that theresulting conjugates (and composition) retain higher bioactivityrelative to PEG conjugates having attachment at Lys-52.

Using this same approach, other conjugates can be prepared usingbranched mPEG2-N-hydroxysuccinimide having other weight averagemolecular weights.

Example 8 Non-Random PEGylation of EPO with mPEG-SMB, 30 kDa

Prior to the conjugation reaction, 1.5 mL of reaction buffer (20 mM-200mM NaPO₄) was added to 500 μL of stock EPO solution. To reversiblyprotect the most reactive amino groups in EPO (e.g., those aminesassociated with the Lys-52 side chain), the EPO solution was adjusted topH 8.0 and was combined with a 10-fold molar excess of dimethylmaleicanhydride (“DMMAn”) relative to the lysine amino acids in EPO, tothereby form a DMMAn-treated EPO solution. The pH was tested andadjusted as necessary to ensure a pH of 8.0.

mPEG-SMB, 30 kDa, stored at −20° C. under argon, was warmed to ambienttemperature. The warmed mPEG-SMB, 30 kDa (30.0 mg) was dissolved in 1.0mL of 2 mM HCl to form an mPEG-SMB solution. The mPEG-SMB solution wasadded to the DMMAn-treated EPO solution (pH 8.0, room temperature),until a thirty molar excess of mPEG-SMB relative to EPO was reached. Toallow for coupling of the mPEG-SMB to EPO via an amide linkage, thereaction solution was stirred for five hours at room temperature andthereafter stirred for nineteen hours at 3-8° C. in a cold room, therebyresulting in a conjugate solution. The reaction was quenched by additionof a 1M glycine solution. Thereafter, to deprotect the protected lysineamino groups, the reaction mixture was adjusted to pH 6.0 with 0.1 N HCland incubated at 37° C. for 30 minutes.

SDS-PAGE was used for characterization. Based upon the SDS-PAGE results,the PEGylation yield of monoPEGylated conjugate (EPO 1-mer) was ˜50%.Based upon the reaction protocol used, it is believed that a majority ofthe conjugate species present in the resulting product mixture possessPEG covalently attached to a site other than the Lys-52, and that theresulting conjugates (and composition) retain higher bioactivityrelative to PEG conjugates having attachment at Lys-52. The productmixture was purified by ion exchange chromatography as previouslydescribed.

The purified monoPEGylated product, designated 01-P-MSMBE-30, wasdetermined by RP-HPLC to contain approximately 91% monomer(mono-mPEG-SMB-30kD-EPO) and 9% dimer (dimPEG-SMB-30kD-EPO). NotriPEGylated product was detected. The purified di-PEGylated product,designated 01-P-DSMBE-30, was determined by RP-HPLC to containapproximately 94% dimer (di-mPEG-SMB-30kD-EPO) and 6% monomer(mono-mPEG-SMB-30kD-EPO). No triPEGylated product was detected.

Preliminary in vivo and in vitro assays were conducted on theconjugates, mono-mPEG-SMB-30kD-EPO and di-mPEG-SMB-30kD-EPO, as providedbelow. These results confirm the bioactivity of the subject conjugates.

TABLE 5 Conjugate In vivo Activity, IU/mg* In vitro Assay**mono-mPEG-SMB- 203000 0.7% 30kD-EPO) Sample 01-P-MSMBE-30di-mPEG-SMB-30kD-EPO 77000 0.4% Sample 01-P-DSMBE-30 *unmodified EPOpossesses an activity of 120,000 IU/mg in the assay set up employed.**for comparison, unmodified EPO is considered to possess an activity of100%.

Using this same approach, other conjugates can be prepared usingmPEG-SMB having other weight average molecular weights.

Example 9 PEGylation of EPO with Branched mPEG-Butyraldehyde, 40 kDa

Branched mPEG2-Butyraldehyde, 40 kDa

PEGylation of EPO was carried out using a branched, N-terminus selectivereagent as shown above.

Branched mPEG2-butyraldehyde, 40 kDa, stored at −20° C. under argon, waswarmed to ambient temperature. The branched mPEG2-butyraldehyde (30.0mg) was dissolved in 2.0 mL of 2 mM HCl to fowl a branchedmPEG2-butyraldehyde solution. The branched mPEG2-butryaldehyde solutionwas added to a previously prepared EPO reaction mixture (500 μL stockEPO solution, 2 mL of 20 mM sodium acetate, pH 5.2) until a twenty molarexcess of branched mPEG2-butryaldehyde to EPO was reached. Afteraddition of the branched mPEG2-butyraldehyde, the pH was tested andadjusted as necessary to ensure a pH of about 6.5. A reducing agent,NaCNBH₃, was added at a ten-fold molar excess relative to the branchedmPEG2-butyraldehyde (with the pH tested and adjusted as necessary toensure a pH of about 6.5). The solution was then stirred for 24 hours at4° C. to ensure coupling via an amine linkage.

The product mixture was analyzed by SDS-PAGE. Based upon the SDS-PAGEresults, the PEGylation yield of the monoPEGylated conjugate (EPO 1-mer)formed by reaction of EPO with branched mPEG2-butyraldehyde was ˜50%. Itis believed that the mixture contained fewer than 50% EPO-conjugatespecies having PEG attached at Lys-52, and fewer than 50% EPO-conjugatespecies having PEG attached at the N-terminal. SDS-PAGE analysis of thepurified product, designated as 01-N-MBC4ALDE-40, demonstrated formationof essentially all, i.e., 100%, monoPEGylated EPO.

The product mixture was purified by ion exchange chromatography aspreviously described. The standard chromatogram of the purifiedconjugate product confirmed production of primarily mono-PEGylatedproduct (˜80%), although a small amount of dimer (˜20%) was detectedusing this technique. SDS-PAGE analysis also indicated that the samplecontained only minimal amounts of higher-PEGylated by-product(s), andconfirmed the near homogeneity of the preparation. MALDI-TOF was used todetermine molecular mass; 01-N-MBC4ALDE-40 was determined to have amolecular mass of approximately 72 kDa, thereby confirmingmono-PEGylation of EPO with the branched mPEG2-butyraldehyde 40 kDreagent.

Using this same approach, other conjugates can be prepared usingbranched mPEG2-butyraldehyde having other weight average molecularweights.

Example 10 PEGylation of EPO with Branched mPEG-Butyraldehyde, 40 kDa

Branched mPEG2-Butyraldehyde, 40 kDa

PEGylation of EPO was carried out using a branched, N-terminus selectivereagent as shown above under slightly different reaction conditions fromthose employed in the preceding example.

Branched mPEG2-butyraldehyde, 40 kDa, stored at −20° C. under argon, waswarmed to ambient temperature. The branched mPEG2-butyraldehyde (30.0mg) was dissolved in 2.0 mL of 2 mM HCl to form a branchedmPEG2-butyraldehyde solution. The branched mPEG2-butryaldehyde solutionwas added to a previously prepared EPO reaction mixture (500 μL stockEPO solution, 2 mL of 20 mM sodium acetate, pH 5.2) until a ten-foldmolar excess of branched mPEG2-butryaldehyde to EPO was reached. Afteraddition of the branched mPEG2-butyraldehyde, the pH was tested andadjusted as necessary to ensure a pH of about 6.5. A reducing agent,NaCNBH₃, was added at a five-fold molar excess relative to the branchedmPEG2-butyraldehyde (with the pH tested and adjusted as necessary toensure a pH of about 6.5). After one hour, another ten-fold molar excessof branched mPEG2-butyraldehyde to EPO was added. Again, the pH wastested and adjusted as necessary to ensure a pH of about 6.5. A secondreducing step was performed by adding NaCNBH₃ at a five-fold molarexcess relative to the second amount of branched mPEG-2 butyraldehydeadded to the solution (with the pH tested and adjusted as necessary toensure a pH of about 6.5). The solution was then stirred for 24 hours at4° C. to ensure coupling via an amine linkage, thereby resulting in aconjugate solution.

The conjugate solution was purified using ion exchange chromatography(anion-strong). SDS-PAGE and RP-HPLC (C₃) analysis were also used forthe characterization. Based upon the purification and analysis, thePEGylation yield of monoPEGylated conjugate (EPO 1-mer) was ˜35%.

The composition is expected to comprise a mixture of monoPEGylatedisoforms having polymer attachment the N-terminal, Lys-52, and otheramines.

Using this same approach, other conjugates can be prepared usingbranched mPEG2-butyraldehyde having other weight average molecularweights.

Example 11 PEGylation of EPO with mPEG-SPA, 20 kDa

mPEG-Succinimidyl propionate having a molecular weight of 20,000 Daltonsis obtained from Nektar Therapeutics, (Huntsville, Ala.). The basicstructure of the PEG reagent is provided below:

To a buffered solution of EPO is added base, e.g., NaOH or an acidequivalent such as HCl, to adjust the pH to approximately 5.5-8.5. Tothe above solution is added a 1.5 to 10-fold molar excess of the PEGreagent, mPEG-SPA, 20 kDa. The resulting mixture is stirred at roomtemperature for several hours. Analysis of the reaction mixture revealssuccessful conjugation of EPO.

Using this same approach, other conjugates can be prepared usingmPEG-SPA having other weight average molecular weights.

Example 12 PEGylation of EPO with mPEG-SMP, 10 kDa

mPEG-SMP having a molecular weight of 10,000 Daltons is obtained fromNektar Therapeutics, (Huntsville, Ala.). The basic structure of the PEGreagent is provided below:

Using this same approach, other conjugates can be prepared usingmPEG-SPA having other weight average molecular weights.

To an aqueous solution of EPO is added a base such as NaOH or an acidequivalent such as HCl to adjust the pH to approximately 5.5-8.5. Tothis solution is then added a 1.5 to 10-fold molar excess of PEGreagent, mPEG-SMP, 10 kDa. The resulting mixture is stirred at roomtemperature for several hours. Analysis of the reaction mixture revealssuccessful conjugation of EPO.

Using this same approach, other conjugates can be prepared usingmPEG-SMP having other weight average molecular weights.

Example 13 PEGylation of Engineered EPO with mPEG-MAL, 20 kDa

Using this same approach, other conjugates can be prepared usingmPEG-SMP having other weight average molecular weights.

mPEG-Maleimide having a molecular weight of 20,000 Daltons is obtainedfrom Nektar Therapeutics, (Huntsville, Ala.). The basic structure of thepolymeric reagent is provided below:

mPEG-MAL, 20 kDa

EPO engineered to include a cysteine residue (see, e.g., Cox et al.,U.S. Pat. No. 6,753,165, or Shaw, et al., WO 90/12874) is stored inbuffer. To this protein solution is added a 3-5 fold molar excess ofmPEG-MAL, 20 kDa. The mixture is stirred at room temperature under aninert atmosphere for several hours. Analysis of the reaction mixturereveals successful conjugation of EPO.

Using this same approach, other conjugates can be prepared usingmPEG-MAL having other weight average molecular weights.

Example 14 PEGylation of Engineered EPO with mPEG-OPSS, 20 kDa

mPEG-OPSS having a molecular weight of 20,000 Daltons is obtained fromNektar Therapeutics, (Huntsville, Ala.). The basic structure of thepolymeric reagent is provided below:

EPO engineered to include a cysteine residue is stored in buffer. Tothis protein solution is added a 3-5 fold molar excess of mPEG-OPSS, 20kDa. The mixture is stirred at room temperature under an inertatmosphere for several hours. Analysis of the reaction mixture revealssuccessful conjugation of EPO.

Using this same approach, other conjugates can be prepared usingmPEG-OPSS having other weight average molecular weights.

Example 15 PEGylation of EPO with mPEG-PIP, 5 kDa

mPEG-PIP having a molecular weight of 5,000 Daltons is obtained fromNektar Therapeutics, (Huntsville, Ala.). The basic structure of thepolymeric reagent is provided below:

A 20-fold molar excess of PEG reagent, mPEG-PIP, 5 kDa, is added to abuffered solution of EPO. The resulting solution is placed on an orbitalshaker set at slow speed to facilitate reaction at room temperature.After 15 minutes, aqueous NaCNBH₃ is added in an amount equal to a 50fold-molar excess relative to EPO. Aliquots are withdrawn at timedintervals from the reaction mixture and are analyzed to determine therate of conjugation. After 24 hours, analysis of the reaction mixturereveals successful conjugation of EPO.

Using this same approach, other conjugates can be prepared usingmPEG-PIP having other weight average molecular weights.

Example 16 In-Vitro Activity of Exemplary PEG-EPO Conjugates

The in-vitro activities of the PEG-EPO conjugates described in thepreceding Examples are determined, e.g., by measuring dose dependentproliferation activities using EPO-responsive target cells, e.g.,primary murine spleen cells (Krystal, G., (1983), Exp. Hematol., 11,649-60), HCD 57, a murine MEL cell line developed by Hankins et al.,(1987), Blood, 70, 173a), and/or UT7-EPO, a human cell line derived fromthe bone marrow of a patient with acute megakaryoblastic leukemia(Komatsu, N., et al., (1991), Cancer Res 51, 341-348).

All of the EPO conjugates described herein for which bioactivity data isnot specifically included are believed to be bioactive.

Example 17 In-Vitro Activity of Certain Exemplary PEG-EPO Conjugates

The biologic activity of the EPO conjugates from Example 3 and Example 9was assessed using a normocythaemic mouse assay (European Pharmacopoia2002). Test dilutions were based on sample concentrations as providedbelow and a rough estimation of a specific potency of 360 kIU/mg. Theconcentrations were determined using the Bradford method (Bradford, M.M., ibid, 1976) or by RP-HPLC by creating a standard curve using eitherBSA (bovine serum albumin) or EPO as the standard.

TABLE 6 Sample Concentrations Example Sample Cross Total DescriptionDesignation Reference Concentration Volume Native EPO NR NR  2.1 mg/ml  1 ml Native control NR NR 0.29 mg/ml   9 ml EPO Mono-mPEG- 01-R-MSMBE-Example 3 0.19 mg/ml 17.4 ml SMB-30kD-EPO 30 Di-mPEG-SMB- 01-R-DSMBE-Example 3 0.17 mg/ml   14 ml 30kD-EPO 30 Mono-branched 01-N- Example 90.203 mg/ml  12.5 ml mPEG2-4- MBC4ALDE- aminobutylene- 40 NH-EPO (frombranched mPEG- 2 butyraldehyde reagent) Native Control NR NR 0.572mg/ml    10 ml EPO

The following reported biological activities represent an approximatedetermination due to a degree of uncertainty regarding the true contentof the PEGylated samples. A first attempt to measure the samples usingtest statistics failed based on a validity level of p>0.95. The data wasrecalculated using p>0.90 and are summarized below.

The assays indicated an increase in biological activity for both the twomonoPEGylated EPO compositions, 01-R-MSMBE-30, and 01-N-MBC4ALDE-40, aswell as the diPEGylated composition, 01-R-DSMBE-30. The observedincrease in biologic activity was similar for each of the samplestested. The calculated biologic activity was 276 kIU/mg for01-R-MSMBE-30, 256 kIU/mg for 01-R-DSMBE-30, and 316 kIU/mg for01-N-MBC4ALDE-40—approximately 2.1 to 2.6 times higher than that of theEPO reference material.

These results demonstrate that PEGylation of EPO to produce EPOconjugates having one or two molecules of these exemplary PEGscovalently attached to EPO results in EPO conjugates having demonstratedbiologic activity.

Example 18 Peptide Mapping of PEG-EPO Conjugates

Peptide mapping was carried out for EPO and various PEG-EPO conjugates,01-R-MSMBE-30 (Example 3), 01-R-DSMBE-30 (Example 3), and01-N-MBC4ALDE-40 (Example 9). Each of the samples was digested withtrypsin, separated by reverse phase chromatography, detected byUV-absorbance, and analyzed by electrospray ionization massspectrometric detection.

FIGS. 1A-D provide total ion current spectra of EPO and the PEG-EPOconjugates described above. The T6-fragment VNFYAWK (SEQ ID NO: 3) andthe T6+T7 fragment VNFYAWKR (SEQ ID NO: 4) contained the lysine atposition 52. The digests of all PEGylated conjugates contained minor orno signals at the retention time corresponding to the tryptic fragmentsT6+T7 (51 minutes) and T6 (63.6 minutes). Due to incomplete digests, apreliminary conclusion regarding the distribution of species in thesamples and their respective sites of PEGylation was not reached.

Additional peptide mapping studies were carried out on the conjugatesdescribed in Example 8. Preliminary results are summarized in the tablebelow.

TABLE 7 01-P-MSMBE-30 01-P-DSMBE-30 (Example 8, Monomer) (Example 8,Dimer) Protein Site % PEGylated % PEGylated Lys 45/52 ~45 ~60 N-terminal~20 ~50 Lys 97/116 ~35 ~55 Lys 20/140/153/155 n.a. n.a.

Example 19 Pharmacokinetic Study in Rats

The mono-mPEG-SMB-30kD-EPO conjugate described in Example 8,01-P-MSMBE-30, was evaluated in a pharmacokinetic study in rats. The invivo bioactivity of the conjugate was determined to be approximately500,000 IU/mg, while its in vitro activity was assessed in preliminaryassays as 1.9%.

Native EPO (used as a control), a marketed version of anerythropoiesis-stimulating protein closely related to EPO, Aranesp®, andmono-mPEG-SMB-30kD-EPO, were used in the comparative study. Proteinswere radiolabeled with ¹²⁵I using the chloramin-T method (Ilondo M M, etal., Biochem Biophys Res Commun, 1986, 134:671-677). Mice were dosedsubcutaneously at 4 μCi/kg body weight. Dosing was conducted on fivesubgroups of four mice each. Blood samples were taken at various timepoints as indicated and analyzed. The results are shown in FIG. 2.

As can be seen, the mono-mPEG-SMB-30kD-EPO conjugate sustains higherblood levels over an extended period of time than either the native EPOcontrol or the marketed product, Aranesp®. Thus, this exemplaryconjugate exhibits a distinctly different pharmacokinetic profile theneither native EPO or Aranesp®, and also possesses a notable advantageover each by virtue of its higher and sustained blood levels over time.

1. A method of preparing a conjugate of erythropoietin (EPO), saidmethod comprising: reacting a polymer comprising the structure:

wherein: POLY is a polyalkylene oxide, Q is an optional linking grouphaving a length of from one to 10 atoms, m is an integer ranging from 0to 20, Z is selected from the group consisting of alkyl, substitutedalkyl, aryl and substituted aryl, and X is a leaving group, witherythropoietin (EPO), under conditions effective to promote reaction ofone or more amino sites on EPO with said polymer to thereby form abiologically active conjugate comprising the structure:

where “—NH-EPO” represents an amino group of EPO.
 2. The method of claim1, wherein said reacting comprises combining an aqueous solution of saidpolymer with an aqueous solution of said EPO to form a polymer-EPOreaction mixture.
 3. The method of claim 2, wherein the pH of saidpolymer-EPO reaction mixture is in a range from about 5 to about
 8. 4.The method of claim 2, wherein said polymer is combined at a 10-fold orgreater molar excess relative to said EPO.
 5. The method of claim 4,wherein said polymer is combined at a 20-fold or greater molar excessrelative to said EPO.
 6. The method of claim 2, wherein said reactingfurther comprises, after said combining, stirring said reaction mixturefor 1-24 hours at a temperature ranging from about −10° C. to about 40°C.
 7. The method of claim 6, wherein said temperature is ambient.
 8. Themethod of claim 1, wherein said method comprises, prior to saidreacting, protecting amino groups of said EPO with a cyclic dicarboxylicacid anhydride protecting agent to form an amino-protected EPO.
 9. Themethod of claim 8, wherein said protecting agent is a maleic orcitraconylic anhydride.
 10. The method of claim 9, wherein saidprotecting agent is dimethylmaleic anhydride.
 11. The method of claim 8,further comprising, after said reacting, deprotecting said amino groupsof said amino-protected EPO.
 12. The method of claim 11, effective toform a conjugate of EPO wherein a minority of said conjugate specieshave a polyalkylene oxide covalently attached at lysine
 52. 13. Themethod of claim 1, further comprising, after said reacting, purifyingsaid conjugate.
 14. The method of claim 1, wherein X is selected fromthe group consisting of chlorine, bromine, N-succinimidyloxy,sulfo-N-succinimidyloxy, 1-benzotriazolyloxy, hydroxyl, 1-imidazolyl,and p-nitrophenyloxy.
 15. The method of claim 1, wherein said polymercomprises the structure:

where n ranges from about 200 to about 1400, and said conjugatecomprises the structure: